Protease inhibitors

ABSTRACT

Compounds of the formula II: 
                         
wherein
         R 1  and R 2  are independently H, F or CH 3 ; or   R 1  forms an ethynyl bond and R 2  is H or C 3 -C 6  cycloalkyl which is optionally substituted with one or two substituents independently selected from methyl, CF 3 , OMe or halo;   R 3  is C 1 -C 3  alkyl or C 3 -C 6  cycloalkyl, either of which is optionally substituted with one or two methyl and/or a fluoro, trifluoromethyl or methoxy, when R 3  is C 3 -C 6  cycloalkyl it may alternatively be gem substituted with fluoro;   R 4  is methyl or fluoro; m is 0, 1 or 2;   E is a bond, or thiazolyl, optionally substituted with methyl or fluoro;   A 1  is CH or N,   A 2  is CR 6 R 7  or NR 6 , provided at least one of A 1  and A 2  comprises N;   R 6  is H, C 1 -C 4  alkyl, C 1 -C 4  haloalkyl, C 1 -C 3  alkyl-O—C 1 -C 3  alkyl, or when A 2  is C, R 6  can also be C 1 -C 4  alkoxy or F;   R 7  is H, C 1 -C 4  alkyl or F
 
or a pharmaceutically acceptable salt, N-oxide or hydrate thereof, have utility in the treatment of disorders characterized by inappropriate expression or activation of cathepsin K, such as osteoporosis, osteoarthritis, rheumatoid arthritis or bone metastases.

This application is a Continuation of application Ser. No. 13/831,267filed on Mar. 14, 2013 now U.S. Pat. No. 8,735,395, which is aContinuation of Ser. No. 13/541,519 filed on Jul. 3, 2012, now U.S. Pat.No. 8,426,421, which is a Divisional of application Ser. No. 12/739,489filed on Oct. 26, 2010, now U.S. Pat. No. 8,242,119 and for whichpriority is claimed under 35 U.S.C. §120. Application Ser. No.12/739,489 is the National Phase of PCT International Application No.PCT/EP2009/062406 filed on Sep. 24, 2009 under 35 U.S.C. §371. Thisapplication also claims priority of Application No. 0817424.5 filed inGreat Britain on Sep. 24, 2008 under 35 U.S.C. §119. The entire contentsof each of the above-identified applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to inhibitors of cysteine proteases, especiallythose of the papain superfamily. The invention provides novel compoundsuseful in the prophylaxis or treatment of disorders stemming frommisbalance of physiological proteases such as cathepsin K.

DESCRIPTION OF THE RELATED ART

The papain superfamily of cysteine proteases is widely distributed indiverse species including mammals, invertebrates, protozoa, plants andbacteria. A number of mammalian cathepsin enzymes, including cathepsinsB, F, H, K, L, O and S, have been ascribed to this superfamily, andinappropriate regulation of their activity has been implicated in anumber of metabolic disorders including arthritis, muscular dystrophy,inflammation, glomerulonephritis and tumour invasion. Pathogeniccathepsin like enzymes include the bacterial gingipains, the malarialfalcipains I, II, III et seq and cysteine proteases from Pneumocystiscarinii, Trypanosoma cruzei and brucei, Crithidia fusiculata,Schistosoma spp.

The inappropriate regulation of cathepsin K has been implicated in anumber of disorders including osteoporosis, gingival diseases such asgingivitis and periodontitis, Paget's disease, hypercalcaemia ofmalignancy and metabolic bone disease. In view of its elevated levels inchondroclasts of osteoarthritic synovium, cathepsin K is implicated indiseases characterised by excessive cartilage or matrix degradation,such as osteoarthritis and rheumatoid arthritis.

It is likely that treatment of bone and cartilage disorders such asosteoarthritis and osteoporosis will require life-long administration ofa cathepsin K inhibitor, often to a patient population within or nearingthe geriatric phase. This places unusually high requirements on the easeof administration of drugs intended for such disorders. For exampleattempts are underway to stretch the dosage regimes of the currentosteoporosis drugs of the bisphosphonate class to weekly or longeradministration regimes to aid compliance. However, even with improveddosing, other side effects of the bisphosphonates remain.Bisphosphonates block bone turnover rather than attenuate it as acathepsin K inhibitor does. For healthy bone it is important to maintainthe remodelling process which bisphosphonates block completely. Inaddition, bis-phosphonates have a very long half-life in bone so ifeffects such as osteonecrosis of the jaw manifest themselves, it isimpossible to remove the bisphosphonate from the bone. In contrast,cathepsin K inhibitors typically have a fast onset and off rate mode ofaction, which means that if a problem was to be identified, dosing couldbe halted and there would be no build up of the inhibitor in the bonematrix.

There is thus a desire for alternative osteoporosis and osteoarthritisdrugs with superior pharmacokinetic and/or pharmacodynamic properties.

International patent application no WO2008/007107 discloses compounds ofthe formula

where Rd is a substituted monocyclic ring, Rc is branched alkyl orcycloalkyl and Ra and Rb are a variety of groups including H, methyl,ethyl, ether, thioether, amine, sulphonate etc. The only compounds whichare prepared have H or methoxy at this position.

There remains a need in the art for potent inhibitors of cathepsin K. Ofparticular benefit are inhibitors of cathepsin K which show selectivityfor cathepsin K over other cathepsins (e.g. selectivity over cathepsin Sand/or cathepsin L). Potent inhibitors of cathepsin K which demonstrateproperties such as high permeability and/or advantageous metabolicprofiles may be expected to be of great value in a clinical setting.Cathepsin K related indications such as osteoporosis or arthritispresuppose protracted periods of administration and therefore it isdesirable that the compounds have minimal toxicity or genotoxicity.

BRIEF DESCRIPTION OF THE INVENTION

According to the present invention, there is provided a compound offormula II:

wherein

-   -   R¹ and R² are independently H, F or CH₃; or    -   R¹ forms an ethynyl bond and R² is H or C₃-C₆ cycloalkyl which        is optionally substituted with one or two substituents        independently selected from methyl, CF₃, OMe or halo;    -   R³ is C₁-C₃ alkyl or C₃-C₆ cycloalkyl, either of which is        optionally substituted with one or two methyl and/or a fluoro,        trifluoromethyl or methoxy, when R³ is C₃-C₆ cycloalkyl it may        alternatively be gem substituted with fluoro;    -   R⁴ is methyl or fluoro; m is 0, 1 or 2;    -   E is thiazolyl, optionally substituted with methyl or fluoro;    -   A₁ is CH or N,    -   A₂ is CR⁶R⁷ or NR⁶, provided at least one of A₁ and A₂ comprises        N;    -   R⁶ is H, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₁-C₃ alkyl-O—C₁-C₃        alkyl, or when A₂ is C, R⁶ can also be C₁-C₄ alkoxy or F;

R⁷ is H, C₁-C₄ alkyl or F

or a pharmaceutically acceptable salt, N-oxide or hydrate thereof.

It will be appreciated that the compounds of the invention can exist ashydrates, such as those of the partial formulae:

and the invention extends to all such alternative forms.

In certain embodiments of the invention, R¹ and R² are both H, bothmethyl or more preferably both F.

In other embodiments of the invention R¹, together with the olefindepicted in Formula II defines an ethynyl bond, for example acetylenewhen R² is H.

In other embodiments of the invention, R¹, together with the olefindepicted in Formula II defines an ethynyl bond and R² is a cycloalkylmoiety, such as cyclopentyl, more preferably cyclobutyl and mostpreferably cyclopropyl. The cycloalkyl is optionally substituted at anyposition, including the 1-position with 1, where valance allows 2substituents selected from methyl, OMe, halo (such as fluoro) or CF₃.

An embodiment of the invention thus defines compounds of the formula:

Where A₂, n, A₁, E, R⁴, m and R³ are as defined above or any of thepreferments defined below and R is H, methyl, CF₃, OMe or halo, such asfluoro, located at any of the 1, 2 or 3 positions.

Suitably R³ is C₁-C₃ alkyl or C₃-C₆ cycloalkyl, either of which isoptionally substituted with one or two methyl and/or a fluoro,trifluoromethyl or methoxy.

Representative values for cycloalkyl for R³ include cyclopropyl,cyclobutyl and especially cyclopentyl or cyclohexyl, any of which beingsubstituted with fluoro or gem fluoro. Gem-fluoro at the 2 position of acyclopropyl, the 3 position of cyclobutyl or cyclopentyl or the 4position of cyclopropyl is often convenient. Gem-fluoro at the 4position of cyclohexyl is also often convenient.

In one embodiment of the invention R³ represents the side chain ofleucine. In a second embodiment of the invention R³ represents the sidechain of isoleucine. In a third embodiment of the invention R³represents the side chain of cyclohexyglycine. In a fourth embodiment ofthe invention R³ represents the side chain of cyclopentylglycine. In afifth embodiment of the invention, R³ represents the side chain ofO-methylthreonine. In a fifth embodiment of the invention R³ representsthe side chain of 4-fluoroleucine. In a sixth embodiment of theinvention R³ represents the side chain of 3-methoxyvaline.

Currently preferred values of R³ include those embodied by the partialstructures:

In one embodiment of the invention m represents 2. Of particularinterest are compounds wherein m represents 1. Still further embodimentsof the invention have m as 0, especially when the adjacent thiazolyl issubstituted with Me or preferably F.

R⁴ suitably represents methyl or fluoro, especially fluoro. If m is 2,it is currently preferred that each R⁴ is the same.

When m represents 1, R⁴ is suitably positioned as shown by the partialstructure:

As defined above, E is thiazolyl, which is optionally substituted withmethyl or more preferably fluoro. The preferred orientation of thethiazolyl ring is:

where R⁵ is H methyl or fluoro.

The ring containing A₁ and A₂ is a saturated, nitrogen-containing ringof 5 or 6 ring atoms. Representative rings thus include pyrrolidin-1-yl,pyrrolidin-3-yl, piperazin-1-yl, piperidin-4-yl and piperidine-1-yl. Thering is conveniently substituted, for example with alkyl or haloalkyl,typically methyl or propyl or trifluoromethyl. Alternatively the ring issubstituted with an ether such as methoxymethyl- or methoxyethyl-. WhenA² is carbon, the ring can alternatively be substituted with alkoxy suchas methoxy, or fluoro, especially gem-fluoro.

A favoured embodiment of the invention has the formula IIa:

wherein

-   -   R¹ and R² are independently H, F or CH₃; or    -   R¹ forms an ethenyl bond and R² is H, C₃-C₆ cycloalkyl,        optionally substituted with one or two substituents selected        from Me, CF₃, OMe or halo (such as fluoro);    -   R³ is branched C₂-C₆ alkyl or C₃-C₆ cycloalkyl, either of which        is substituted with halo or trifluoromethyl;    -   R⁴ is methyl or fluoro; m is 0 or 1 or 2;    -   R⁵ is H, methyl or fluoro;    -   R⁶ is C₁-C₄ alkyl;        or a pharmaceutically acceptable salt, N-oxide or hydrate        thereof (collectively referred to herein as compounds of the        invention).

R⁵ is preferably fluoro, especially when m is 0. The remainingpreferments are as defined above in relation to Formula II. Referencesto formula II below are understood to include the correspondingembodiments of formula IIa.

Representative embodiments of formula II, in which the —C═CR¹R² moietyis acetylene include:

-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide    and pharmaceutically acceptable salts, N-oxides and hydrates    thereof.

Further representative embodiments of formula II, in which R¹ and R² aremethyl include:

-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methylbutyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(dimethylvinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide    and pharmaceutically acceptable salts, N-oxides and hydrates    thereof.

Further preferred embodiments of formula II, in which R¹ and R² are Finclude

-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-m    ethyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-m    ethyl-butyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(difluorovinyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide    and pharmaceutically acceptable salts, N-oxides and hydrates    thereof.

Further preferred embodiments of formula II, in which R¹ is an ethynylbond and R² is cyclopropyl include:

-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-3-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclohexyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide;-   N-[1-(6-(cyclopropylethynyl)-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-carbonyl)-2-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide    and pharmaceutically acceptable salts, N-oxides and hydrates    thereof.

The C₁-C_(n) alkyl definition of R⁶ or R⁷ is meant to include bothbranched and unbranched alkyl moieties containing between one and ncarbon atoms in total. Examples of such R⁶ groups or R⁷ are methyl,ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl,tert-butyl and sec-butyl). One R⁶ group of particular interest ismethyl. A second R⁶ group of particular interest is propyl (especiallyn-propyl). As R³ is optionally substituted with one or two methylgroups, this moiety may also define a branched alkyl chain of up to 5 Catoms.

In some embodiments of the invention A₁ is N.

Additional aspects of the invention include a pharmaceutical compositioncomprising a compound as defined above and a pharmaceutically acceptablecarrier or diluent therefor.

A further aspect of the invention is the use of a compound as definedabove in the manufacture of a medicament for the treatment of disordersmediated by cathepsin K, such as:

-   -   osteoporosis,    -   gingival diseases (such as gingivitis and periodontitis),    -   Paget's disease,    -   hypercalcaemia of malignancy,    -   metabolic bone disease,    -   diseases characterised by excessive cartilage or matrix        degradation (such as osteoarthritis and rheumatoid arthritis),    -   bone cancers including neoplasia,    -   pain (especially chronic pain).

Additionally provided is a method for the treatment or prevention of adisorder mediated by cathepsin K comprising the administration of a safeand effective amount of a compound of the invention for the purpose oftreating or preventing said disorder which is mediated by cathepsin K.

Also provided is a compound of the invention for the treatment orprevention of a disorder mediated by cathepsin K.

Further, there is provided as an aspect of the invention novelintermediates (as described herein) which may be of use in thepreparation of the compounds of the invention.

In particular there is provided a compound of the formula:

or an N-protected derivative thereof (e.g. Boc, CBz, or Fmoc-protected).Also provided by the invention is the corresponding 1,3-dioxolaneprotected analogue and N-protected derivatives thereof (e.g. Boc-CBz, orFmoc protected). Particularly favoured embodiments of this buildingblock have both R¹ and R² as fluoro, or both methyl or the —C═C(R¹)R²moiety is acetylene or cyclopropylethylene.

A further novel intermediate of the invention is has the formula:

or the corresponding 1,3-dioxolane protected analogue, in each casewherein the N function is optionally protected with a conventionalprotecting group such as Boc, CBz or Fmoc.

The compounds of the invention can form salts which form an additionalaspect of the invention. Appropriate pharmaceutically acceptable saltsof the compounds of Formula II include salts of organic acids,especially carboxylic acids, including but not limited to acetate,trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,malate, pantothenate, isethionate, adipate, alginate, aspartate,benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate,glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate,palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate,tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate,organic sulphonic acids such as methanesulphonate, ethanesulphonate,2-hydroxyethane sulphonate, camphorsulphonate, 2-napthalenesulphonate,benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate;and inorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoricand sulphonic acids.

The compounds of the invention may in some cases be isolated as thehydrate. Hydrates are typically prepared by recrystallisation from anaqueous/organic solvent mixture using organic solvents such as dioxin,tetrahydrofuran or methanol. Hydrates can also be generated in situ byadministration of the corresponding ketone to a patient.

The N-oxides of compounds of the invention can be prepared by methodsknown to those of ordinary skill in the art. For example, N-oxides canbe prepared by treating an unoxidized form of the compound of theinvention with an oxidizing agent (e.g., trifluoroperacetic acid,permaleic acid, perbenzoic acid, peracetic acid,meta-chloroperoxybenzoic acid, or the like) in a suitable inert organicsolvent (e.g., a halogenated hydrocarbon such as dichloromethane) atapproximately 0° C. Alternatively, the N-oxides of the compounds of theinvention can be prepared from the N-oxide of an appropriate startingmaterial.

Examples of N-oxides of the invention include those with the partialstructures:

Compounds of the invention in unoxidized form can be prepared fromN-oxides of the corresponding compounds of the invention by treatingwith a reducing agent (e.g., sulfur, sulfur dioxide, triphenylphosphine, lithium borohydride, sodium borohydride, phosphorusbichloride, tribromide, or the like) in an suitable inert organicsolvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0to 80° C.

It should be noted that the radical positions on any molecular moietyused in the definitions may be anywhere on such moiety as long as it ischemically stable.

Radicals used in the definitions of the variables include all possibleisomers unless otherwise indicated. For instance butyl includes t-butyl,i-butyl, n-butyl etc.

When any variable occurs more than one time in any constituent, eachdefinition is independent.

Unless otherwise mentioned or indicated, the chemical designation of acompound encompasses the mixture of all possible stereochemicallyisomeric forms, which said compound may possess. Said mixture maycontain all diastereomers and/or enantiomers of the basic molecularstructure of said compound. All stereochemically isomeric forms of thecompounds of the present invention both in pure form or mixed with eachother are intended to be embraced within the scope of the presentinvention.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible isomers) up to a stereoisomericexcess of 100% (i.e. 100% of one isomer and none of the other), more inparticular, compounds or intermediates having a stereoisomeric excess of90% up to 100%, even more in particular having a stereoisomeric excessof 94% up to 100% and most in particular having a stereoisomeric excessof 97% up to 100%. The terms “enantiomerically pure” and“diastereomerically pure” should be understood in a similar way, butthen having regard to the enantiomeric excess, and the diastereomericexcess, respectively, of the mixture in question.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomer. While resolution of enantiomers can be carried outusing covalent diasteromeric derivatives of compounds of Formula II,dissociable complexes are preferred (e.g., crystalline;diastereoisomeric salts). Diastereomers have distinct physicalproperties (e.g., melting points, boiling points, solubilities,reactivity, etc.) and can be readily separated by taking advantage ofthese dissimilarities. The diastereomers can be separated bychromatography, for example HPLC or, preferably, byseparation/resolution techniques based upon differences in solubility.The optically pure enantiomer is then recovered, along with theresolving agent, by any practical means that would not result inracemization. A more detailed description of the techniques applicableto the resolution of stereoisomers of compounds from their racemicmixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen,Enantiomers, Racemates and Resolutions, John Wiley & Sons, Inc. (1981).

The compounds of formula II or any subgroup of formula II as definedherein include radioisotopes or radiomarked compounds, wherein one ormore of the atoms is replaced by an isotope of that atom, i.e. an atomhaving the same atomic number as, but an atomic mass different from, theone(s) typically found in nature. Examples of isotopes that may beincorporated into the compounds of formula I or any subgroup of formulaI, include but are not limited to isotopes of hydrogen, such as ²H and³H (also denoted D for deuterium and T for tritium respectively),carbon, such as ¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen,such as ¹⁵C, ¹⁷C and ¹⁸C, phosphorus, such as ³¹P and ³²P, sulphur, suchas ³⁵5, fluorine, such as ¹⁸F, chlorine, such as ³⁶Cl, bromine such as⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³I, ¹²⁴I, ¹²⁵I and¹³¹I.

The choice of isotope included in an isotope-labelled compound willdepend on the specific application of that compound. For example, fordrug or substrate tissue distribution assays, compounds wherein aradioactive isotope such as ³H or ¹⁴C is incorporated will generally bemost useful. For radio-imaging applications, for example positronemission tomography (PET) a positron emitting isotope such as ¹¹C, ¹⁸F,¹³N or ¹⁵O will be useful. The incorporation of a heavier isotope, suchas deuterium, i.e. ²H, may provide greater metabolic stability to acompound of formula I or any subgroup of formula I, which may result in,for example, an increased in vivo half life of the compound or reduceddosage requirements.

For synthetic convenience it will generally be preferred that thecompounds of formula II are in the natural isotopic state.

Isotopically labelled compounds of formula I or any subgroup of formulaII can be prepared by processes analogous to those described in theSchemes and/or Examples herein below by using the appropriateisotopically labelled reagent or starting material instead of thecorresponding non-isotopically labelled reagent or starting material, orby conventional techniques known to those skilled in the art.

It will be appreciated that the invention extends to prodrugs, solvates,complexes and other forms releasing a compound of the invention in vivo.

While it is possible for the active agent to be administered alone, itis preferable to present it as part of a pharmaceutical formulation.Such a formulation will comprise the above defined active agent togetherwith one or more acceptable carriers/excipients and optionally othertherapeutic ingredients. The carrier(s) must be acceptable in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration, but preferably the formulation is an orally administeredformulation. The formulations may conveniently be presented in unitdosage form, e.g. tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the abovedefined active agent with the carrier. In general, the formulations areprepared by uniformly and intimately bringing into association theactive agent with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product. The invention extendsto methods for preparing a pharmaceutical composition comprisingbringing a compound of Formula II or its pharmaceutically acceptablesalt in conjunction or association with a pharmaceutically acceptablecarrier or vehicle. If the manufacture of pharmaceutical formulationsinvolves intimate mixing of pharmaceutical excipients and the activeingredient in salt form, then it is often preferred to use excipientswhich are non-basic in nature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may bepresented as discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active agent; as a powder orgranules; as a solution or a suspension of the active agent in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water in oil liquid emulsion and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets andcapsules), the term suitable carrier includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropyl-methylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring or the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally be coated or scored and may be formulatedso as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

The appropriate dosage for the compounds or formulations of theinvention will depend upon the indication and the patient and is readilydetermined by conventional animal trials. Dosages providingintracellular (for inhibition of physiological proteases of the papainsuperfamily) concentrations of the order 0.01-100 uM, more preferably0.01-10 uM, such as 0.1-25 uM are typically desirable and achievable.

Compounds of the invention are prepared by a variety of solution andsolid phase chemistries.

The compounds are typically prepared as building blocks reflecting theP1, P2 and P3 moieties of the end product inhibitor. Without in any waywishing to be bound by theory, or the ascription of tentative bindingmodes for specific variables, the notional concepts P1, P2 and P3 asused herein are provided for convenience only and have substantiallytheir conventional Schlecter & Berger meanings and denote those portionsof the inhibitor believed to fill the S1, S2, and S3 subsitesrespectively of the enzyme, where S1 is adjacent the cleavage site andS3 remote from the cleavage site. Compounds defined by Formula II areintended to be within the scope of the invention, regardless of bindingmode.

Broadly speaking the P1 building block will have the formula:

whereinR¹ and R² are as defined above, the two Rb groups define a ketal, suchas the bis methyl ketal or together define a cyclic ketal such as1,3-dioxolane;and Rc is an hydroxy protecting group. Less commonly Rc is H orrepresents the keto function of the end-product inhibitor in cases wherethe P1 building block as the ketone is elongated with P2 and P3.

WO05/066180 describes the preparation of intermediates towards the aboveP1 building blocks, including:

The first stage in the synthesis of compounds of the invention istypically the preparation in solution of a functionalized P1 buildingblock. Scheme 1 illustrates a route to a convenient 6-aldehydeintermediate.

The starting bicyclic alcohol (1a) can be prepared as described inWO05/066180. Oxidation of the hydroxy function for example withDess-Martin periodinane followed by transformation of the afforded ketofunction into a dimethyl ketal effected by treatment with trimethylorthoformate in the presence of an acid like p-toluenesulphonic acidprovides the ketal (1b). Removal of the Cbz and benzyl protecting groupseffected for instance by hydrogenolysis using a catalyst like Pd(OH)₂ orthe like, followed by boc protection of the afforded free amine providesthe alcohol (1c). Oxidation of the afforded free alcohol using forinstance Dess-Martin periodinane in a solvent like dichloromethanefollowed by a Wittig reaction using methyl triphenylphosphinium bromidein the presence of KOt.Bu or the like provides the olefin (1d).Hydroxylation of the double bond effected for example by treatment with9-BBN—H, provides the primary alcohol (1e) which subsequently can beoxidized to the corresponding aldehyde (1f) using any suitable oxidationmethod such as treatment with Dess-Martin periodinane or the like.

Scheme 2 illustrates a typical procedure for a 6-acetylene P1 buildingblock commencing from the 6-aldehyde intermediate of Scheme 1.

The C-6 acetylene building block 1 h is prepared as shown in Scheme 2.Essentially, the acetylene 1 h is readily prepared from the aldehydeprecursor if by the two step Corey-Fuchs methodology via thecorresponding 1,1-dibromoolefin 1g. Aldehyde if is treated withtriphenylphosphinecarbondibromide, for example generated from Ph₃PCH₂Cl₂and CBr₄, at 0° C. to afford the dibromoalkene 1g. Subsequent treatmentof the dibromide in an organic solvent such as THF at reducedtemperature, typically −78° C. with n-butyl lithium affords on work-up,the acetylene 1h, generally in good yield. A number of conventionalN-protecting groups could be used instead of the Boc group shown here.In addition, the dimethylketal can be replaced by a number of standardketone protecting groups including cyclic ketals or, less commonly, thebuilding block could be built up with the active keto functionality inplace, prior to coupling.

Compounds of formula II, where R¹ and/or R² define a fluorovinylsubstituent are prepared by adding a fluoromethyl halide, such asdibromodifluoromethane to a solution of triphenylphosphine in an organicsolvent such as DMF for example at 0° C. under an inert atmosphere. Thesolution is stirred for a short period of time (such 0.5 hrs) and thenthe aldehyde if and zinc powder are slowly added. After an hour at roomtemperature the reaction can be quenched by addition of aqueous NaHCO₃to yield a protected P1 building block 1h′ (represented above the N-Bocand dimethyl acetal, but other conventional protecting groups can beenvisaged).

Compounds of formula II, wherein R¹ and/or R² define a methylated vinylsubstituent can be prepared by a Wittig reaction. For example, potassiumtert-butoxide is added to a suspension of an activated alkyl, such asisopropyl triphenylphosphonium iodide in diethyl ether. After approx. 2hrs aldehyde 1f, dissolved in a solvent such as diethylether is added tothe solution. After stirring (eg 1 hr) the reaction can be quenched withaqueous ammonium chloride to yield the vinyl building block 1h″(illustrated above as the dimethylvinyl). Although the scheme has beendepicted with the N-Boc and dimethyl acetal protecting groups, it willbe apparent that other conventional protecting groups can be envisaged.

Compounds of formula II wherein R² is an optionally substitutedcycloalkyl group may be prepared by alkylation of the correspondingethynyl building block. In the example above 1 h is treated with astrong base, for example sodium amide, to get the acetylide, which issubsequently reacted with the appropriate cycloalkyl halide to yield thebuilding block 1h′″ (in the example above cyclopropyl chloride, butother activated cycloalkyls will also be effective). Alternatively theacetylene function can be extended by reaction with ClCH₂CH₂Br followedby treatment with a base to form a cyclopropyl ring. Although the schemehas been depicted with the N-Boc and dimethyl acetal protecting groups,it will be apparent that other conventional protecting groups can beenvisaged.

Typically to get to the final compound, the appropriate P1 buildingblocks such 1h, 1h′, 1h″ or 1h′″, is N-deprotected in a conventionalfashion, such as treatment with acetyl chloride in methanol to remove anN-Boc protecting group. With the subsequent free amine, the P2 residueis introduced, eg via BocP2-OH using standard coupling conditions suchas HATU, DIPEA in DMF. The terminal Boc protection is again removed withacetyl chloride in methanol and the P3 residue introduced via P3-OHusing standard coupling conditions such as HATU, DIPEA in DMF. Finallythe dimethylketal protection is removed with TFA to afford the requiredfinal compound.

Elongation is typically carried out in the presence of a suitablecoupling agent e.g., benzotriazole-1-yloxytrispyrrolidinophosphoniumhexafluorophosphate (PyBOP),O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or1,3-dicyclohexyl carbodiimide (DCC), optionally in the presence ofl-hydroxybenzotriazole (HOBT), and a base such asN,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and thelike. The reaction is typically carried out at 20 to 30° C., preferablyat about 25° C., and requires 2 to 24 h to complete. Suitable reactionsolvents are inert organic solvents such as halogenated organic solvents(e.g., methylene chloride, chloroform, and the like), acetonitrile,N,N-dimethylformamide, ethereal solvents such as tetrahydrofuran,dioxane, and the like.

Alternatively, the above elongation coupling step can be carried out byfirst converting the P3/P2 building block into an active acid derivativesuch as succinimide ester and then reacting it with the P1 amine. Thereaction typically requires 2 to 3 h to complete. The conditionsutilized in this reaction depend on the nature of the active acidderivative. For example, if it is an acid chloride derivative, thereaction is carried out in the presence of a suitable base (e.g.triethylamine, diisopropylethylamine, pyridine, and the like). Suitablereaction solvents are polar organic solvents such as acetonitrile,N,N-dimethylformamide, dichloromethane, or any suitable mixturesthereof.

The P2 building block is typically an N-protected amino acid such asL-leucine, L-isoleucine, O-methyl-L-threonine, L-3-hydroxyvaline,4-fluoroleucine, L-cyclopentylglycine or L-cyclohexylglycine, and P3typically comprises a capping group such as a benzoic acid derivativewith, eg, the N-alkyl-piperazinyl-E moiety already introduced orprovided with a synthon therefor in the para position.

The suitably protected individual building blocks can first be preparedand subsequently coupled together, preferably in the sequenceP2+P1→P2−P1 followed by N-alkylpiperazinyl-E-benzoicacid*+P2−P1→N-alkylpiperazinyl-E-benzoate-P2−P1, where * denotes anactivated form, in order to minimise racemisation at P2.

Coupling between two amino acids, an amino acid and a peptide, or twopeptide fragments can be carried out using standard coupling proceduressuch as the azide method, mixed carbonic-carboxylic acid anhydride(isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide,diisopropylcarbodiimide, or water-soluble carbodiimide) method, activeester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method,Woodward reagent K-method, carbonyldiimidazole method, phosphorusreagents or oxidation-reduction methods. Some of these methods(especially the carbodiimide method) can be enhanced by adding1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can beperformed in either solution (liquid phase) or solid phase.

More explicitly, the coupling step involves the dehydrative coupling ofa free carboxyl of one reactant with the free amino group of the otherreactant in the present of a coupling agent to form a linking amidebond. Descriptions of such coupling agents are found in generaltextbooks on peptide chemistry, for example, M. Bodanszky, “PeptideChemistry”, 2nd rev ed., Springer-Verlag, Berlin, Germany, (1993)hereafter simply referred to as Bodanszky, the contents of which arehereby incorporated by reference. Examples of suitable coupling agentsare N,N′-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole in thepresence of N,N′-dicyclohexylcarbodiimide orN-ethyl-N′-[(3-dimethylamino) propyl]carbodiimide. A practical anduseful coupling agent is the commercially available(benzotriazol-1-yloxy)tris-(dimethylamino) phosphoniumhexafluorophosphate, either by itself or in the present of1-hydroxybenzotriazole or 4-DMAP. Another practical and useful couplingagent is commercially available2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.Still another practical and useful coupling agent is commerciallyavailable O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate.

The coupling reaction is conducted in an inert solvent, e.g.dichloromethane, acetonitrile or dimethylformamide. An excess of atertiary amine, e.g. diisopropylethylamine, N-methylmorpholine,N-methylpyrrolidine or 4-DMAP is added to maintain the reaction mixtureat a pH of about 8. The reaction temperature usually ranges between 0°C. and 50° C. and the reaction time usually ranges between 15 min and 24h.

The functional groups of the constituent non-natural amino acidsgenerally must be protected during the coupling reactions to avoidformation of undesired bonds. The protecting groups that can be used arelisted in Greene, “Protective Groups in Organic Chemistry”, John Wiley &Sons, New York (1981) and “The Peptides: Analysis, Synthesis, Biology”,Vol. 3, Academic Press, New York (1981), hereafter referred to simply asGreene, the disclosures of which are hereby incorporated by reference.

The alpha-carboxyl group of the C-terminal residue is usually protectedas an ester that can be cleaved to give the carboxylic acid. Protectinggroups that can be used include 1) alkyl esters such as methyl,trimethylsilyl and t-butyl, 2) aralkyl esters such as benzyl andsubstituted benzyl, or 3) esters that can be cleaved by mild base ormild reductive means such as trichloroethyl and phenacyl esters.

The alpha-amino group of each amino acid to be coupled is typicallyN-protected. Any protecting group known in the art can be used. Examplesof such groups include: 1) acyl groups such as formyl, trifluoroacetyl,phthalyl, and p-toluenesulfonyl; 2) aromatic carbamate groups such asbenzyloxycarbonyl (Cbz or Z) and substituted benzyloxycarbonyls, and9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate groups suchas tertbutyloxycarbonyl (Boc), ethoxycarbonyl,diisopropylmethoxy-carbonyl, and allyloxycarbonyl; 4) cyclic alkylcarbamate groups such as cyclopentyloxycarbonyl andadamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl andbenzyl; 6) trialkylsilyl such as trimethylsilyl; and 7) thiol containinggroups such as phenylthiocarbonyl and dithiasuccinoyl. The preferredalpha-amino protecting group is either Boc or Fmoc. Many amino acidderivatives suitably protected for peptide synthesis are commerciallyavailable.

The alpha-amino protecting group is typically cleaved prior to the nextcoupling step. When the Boc group is used, the methods of choice aretrifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane orin ethyl acetate. The resulting ammonium salt is then neutralized eitherprior to the coupling or in situ with basic solutions such as aqueousbuffers, or tertiary amines in dichloromethane or acetonitrile ordimethylformamide. When the Fmoc group is used, the reagents of choiceare piperidine or substituted piperidine in dimethylformamide, but anysecondary amine can be used. The deprotection is carried out at atemperature between 0° C. and room temperature usually 20-22° C.

Once the inhibitor sequence is completed any remaining protecting groupsare removed in whatever manner is dictated by the choice of protectinggroups. These procedures are well known to those skilled in the art.

P2 building blocks in the form of N-protected L-amino acids are readilyavailable commercially, for example L-leucine, L-isoleucine,L-cyclohexylglycine, O-methyl-L threonine and others are availablecommercially with a number of protecting group variants such as CBz, Bocor Fmoc. Other variants of R³ are easily prepared from commerciallyavailable starting materials. For example compounds wherein R³ is—C(CH₃)₂OCH₃ can be prepared by reacting CBz protected(S)-(+)-2-amino-3-hydroxy-3-methylbutanoic acid with3,3-dimethoxy-hexahydro-furo(3,2b)pyrrole to form the desired P2−P1unit. The P2 side chain alcohol can now be methylated using methyliodideunder conventional sodium hydride, imidazole, THF conditions to obtainthe desired P2 without substantial racemisation of the alpha centre.This P2−P1 moiety can now be carried through the synthesis as describedherein, namely CBz removal and coupling.

WO05/565299 describes the preparation of a gamma-fluoroleucine P2building block. An alternative synthesis of Fmoc andN-Boc-gammafluoroleucine building blocks is shown in Truong et al Syn.Lett. 2005 no 8 1278-1280.

The preparation of P3 building blocks are described in WO05/066180,WO08/007,114 or readily prepared by analogous methods. For example,Scheme E below shows the preparation of a P3 building block wherein E isa fluoro-substituted thiazolyl:

Synthesis of4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]benzoic acid

The starting material, methyl 4-acetylbenzoate, is commerciallyavailable. Bromination at the α-position to the ketone is achieved withbromine in acetic acid to provide the desired 4-(2-bromo-acetyl)-benzoicacid methyl ester. Subsequent treatment of 4-(2-bromo-acetyl)-benzoicacid methyl ester with potassium fluoride in the presence of 18-crown-6at 90° C., provides 4-(2-fluoro-acetyl)-benzoic acid methyl ester aftercolumn chromatography. Repeated bromination at the α-position to theketone is achieved with bromine in acetic acid to provide the desired4-(2-bromo-2-fluoro-acetyl)-benzoic acid methyl ester. Formation of thethiazole is typically carried out by heating4-(2-bromo-2-fluoro-acetyl)-benzoic acid methyl ester with4-methylpiperazine-1-carbothioamide at 70° C. for 2 hours. On cooling,the desired4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acidmethyl ester precipitates out. Deprotection of the methyl ester iscarried out using a lithium hydroxide solution and the desired acid,4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acid isgenerally obtained in good yield as the dihydrochloride salt on workupwith hydrochloric acid.

The term “N-protecting group” or “N-protected” as used herein refers tothose groups intended to protect the N-terminus of an amino acid orpeptide or to protect an amino group against undesirable reactionsduring synthetic procedures. Commonly used N-protecting groups aredisclosed in Greene, “Protective Groups in Organic Synthesis” (JohnWiley & Sons, New York, 1981), which is hereby incorporated byreference. N-protecting groups include acyl groups such as formyl,acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl,o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such asbenzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forminggroups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl andthe like; and silyl groups such as trimethylsilyl and the like. FavouredN-protecting groups include formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) andbenzyloxycarbonyl (Cbz).

Hydroxy and/or carboxy protecting groups are also extensively reviewedin Greene ibid and include ethers such as methyl, substituted methylethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such astrimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl,triphenylsilyl, t-butyldiphenylsilyl triisopropyl silyl and the like,substituted ethyl ethers such as 1-ethoxymethyl,1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl,dipehenylmethyl, triphenylmethyl and the like, aralkyl groups such astrityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especiallythe chloride). Ester hydroxy protecting groups include esters such asformate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate,pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonatehydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyland the like.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various embodiments of the invention will now be described by way ofillustration only with reference to the following Examples and FIG. 1which depicts plasma concentration against time for male C57Bl miceorally administered a compound of the invention or a compound of theprior art.

Reference Example 1 A P3 Building Block Step a) 4-Cyanopropiophenone

As described for the preparation of 4-cyanoacetophenone (Synth. Commun1994, 887-890), a mixture of 4-bromopropiophenone (5.65 g, 26.4 mmol),Zn(CN)₂ (1.80 g, 15.3 mmol), and Pd(PPh₃)₄ (2.95 g, 2.6 mmol) wasrefluxed at 80° C. in deoxygenated DMF (35 mL, stored over 4 Å molecularsieves, bubbled with Ar before use) for 18 h. The mixture waspartitioned between toluene (100 mL) and 2N NH₄OH (100 mL). The organicphase was extracted with 2N NH₄OH (100 mL), washed with saturatedaqueous NaCl (2×100 mL), dried, and evaporated. A 10 mmol scale reactionwas done similarly and the crude products were combined. Flashchromatography (330 g silica, 6/1 petroleum ether-EtOAc) gave whitesolids (5.17 g, 89%).

1H NMR (CDCl₃) δ ppm: 1.22 (t, 3H, J=7.2 Hz), 3.00 (q, 2H, J=7.3 Hz),7.75 (d, 2H, J=8.8 Hz), 8.03 (d, 2H, J=8.4 Hz)

13C NMR (CDCl₃) δ ppm: 7.8, 32.1, 116.1, 117.9, 128.3, 132.4, 139.7,199.2

Step b) 4-Propionylbenzoic Acid

4-Cyanopropiophenone (4.67 g, 29.3 mmol) was refluxed with 2N NaOH (90mL, 180 mmol) and dioxane (90 mL) at 95° C. overnight. The mixture wasdiluted with water (150 mL), washed with ether (75 mL), acidified to pH2 with concentrated HCl, and extracted with ether (3×75 mL). The organicphase was washed with saturated aqueous NaCl (3×75 mL), dried, andevaporated to give yellow solids (5.12 g, 98%).

1H NMR (CDCl₃+CD₃OD) δ ppm: 1.18 (t, 3H, J=7.2 Hz), 2.99, (q, 2H, J=7.1Hz), 7.95 (d, 2H, J=8.4 Hz), 8.08 (d, 2H, J=8.8 Hz)

13C NMR (CDCl₃) δ ppm: 7.9, 32.1, 127.7, 130.0, 134.0, 140.0, 168.0,200.8

Step c) Methyl 4-Propionylbenzoate

The benzoic acid above (890 mg, 5 mmol), NaHCO₃ (1.26 g, 15 mmol) andiodomethane (935 μL, 15 mmol) in DMF (10 mL) were stirred at RTovernight. The mixture was diluted with saturated aqueous NaCl (50 mL)and extracted with ether (3×50 mL). The organic phase was washed withwater (50 mL), dried, and evaporated. Flash chromatography (90 g silica,2/1 petroleum ether-EtOAc) gave white solids (744 mg, 77%).

1H NMR (CDCl₃) δ ppm: 1.24 (t, 3H, J=7 Hz), 3.03 (q, 2H, J=7 Hz), 3.95(s, 3H), 8.0 and 8.12 (ABq, 4H)

Step d) Methyl 4-(2-Bromopropionyl)Benzoate

Methyl 4-propionylbenzoate (744 mg, 3.87 mmol), pyrrolidonehydrotribromide (1.98 g), and 2-pyrrolidinone (380 mg, 4.5 mmol) in THF(38 mL) were heated at 50° C. under nitrogen for 3 h. The mixture wascooled, filtered, concentrated, and then redissolved in ether (50 mL).The ether solution was washed successively with water (20 mL), saturatedaqueous Na₂S₂O₅ (20 mL), saturated aqueous NaCl (20 mL), and water (20mL), dried and evaporated to give a yellow oil (1.025 g) that was useddirectly in the Hantzsch coupling. This material contained 91% of thedesired bromoketone, 5% starting ketone, and 4% 4-bromo-1-butanol, asdetermined by 1H NMR.

1H NMR (CDCl₃) δ ppm: 1.92 (d, 3H, J=7 Hz), 3.96 (s, 3H), 5.28 (q, 1H,J=7 Hz), 8.07 and 8.14 (ABq, 4H)

Step e)4-[2-(4-tert-Butoxycarbonylpiperazin-1-yl)-5-methylthiazol-4-yl]benzoicacid methyl ester

All of the α-bromoketone above and4-thionocarbonylpiperazine-1-carboxylic acid tert-butyl ester (J. Med.Chem., 1998, 5037-5054, 917 mg, 3.73 mmol) were refluxed in 36 mL THF at70° C. for 2 h, under N₂. The precipitate was filtered and the filtrateevaporated to give yellow solids. Flash column chromatography (silica,5/1 petroleum ether-EtOAc) gave 624 mg of light yellow solids.Chromatography of the precipitate (silica, 2/1 petroleum ether-EtOAc)gave 32 mg more of compound. Total yield is 44%.

1H NMR (CDCl₃) δ ppm: 1.46 (s, 9H), 2.43 (s, 3H), 3.42, (m, 4H), 3.54(m, 4H), 3.90 (s, 3H), 7.68 and 8.04 (ABq, 4H).

Step f)4-[2-(4-tert-Butoxycarbonylpiperazin-1-yl)-5-methylthiazol-4-yl]benzoicacid

The above methyl ester (564 mg, 1.35 mmol) was heated with 1.35 mL 2NNaOH, 5 mL THF, and 3.65 mL water at 60° C. for 4 h. The reactionmixture was evaporated, poured into 20 mL saturated aqueous NaCl and 20mL CH₂Cl₂, and then acidified to pH 3 with 5% citric acid, in an icebath. The layers were separated and the organic phase was extractedfurther with 2×10 mL CH₂Cl₂. The organic phases were combined, washedwith water (10 mL), dried, and evaporated to give light yellow solids(537 mg, 98%).

1H NMR (CDCl₃) δ ppm: 1.48 (s, 9H), 2.47 (s, 3H), 3.47 (m, 4H), 3.57 (m,4H), 7.74 and 8.12 (ABq, 4H).

13C NMR (CDCl₃) δ ppm: 12.6, 28.3, 42.8, 48.1, 80.3, 119.1, 127.8,128.2, 130.1, 140.5, 145.6, 154.6, 167.2, 171.4.

LCMS: (M+H)⁺ 404, (M−H)⁻ 402.

Step g) 4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]benzoicacid

4-[4-(4-Carboxy-phenyl)-5-methyl-thiazol-2-yl]piperazine-1-carboxylicacid tert-butyl ester (0.421 mmol) was dissolved in 4M HCl in1,4-dioxane, and stirred at room temperature for 1 h. The solvent wasthen removed under vacuum, and the residue4-(5-methyl-2-piperazin-1-yl-thiazol-4-yl)-benzoic acid was suspended inmethanol (10 ml) and treated with AcOH/AcONa buffer (pH ˜5.5, 5 ml), andformaldehyde (0.547 mmol). The reaction mixture was stirred at roomtemperature for 1 h, then treated with NaCNBH₃ (0.547 mmol) and stirredat room temperature overnight. The solvent was then removed undervacuum, and the residue was purified by column chromatography to affordthe title compound (0.403 mmol, 95%).

MS (ES) m/z 318 (100%, [M+H]⁺).

Reference Example 2 An Alternative P3 Building Block3-Fluoro-4-[2-(4-methylpiperazin-1-yl)-thiazol-4-yl]benzoic acid HClsalt

Step a) Methyl 4-bromo-3-fluorobenzoate

4-Bromo-3-fluorobenzoic acid (2.46 g, 11.2 mmol) was dissolved in MeOH(9 mL) and toluene (4 mL) and cooled in an ice bath.(Trimethylsilyl)diazomethane (11 mL, 2.0 M in hexanes, 22 mmol) wasadded dropwise until the yellow color persisted. The solution wasstirred at room temperature for 40 mins and then concentrated in vacuo.A second batch of carboxylic acid (2.43 g) was treated similarly. Thecrude product from both batches were combined and subjected to flashchromatography (silica, 5/1 pentane-EtOAc) to give the methyl ester aswhite solids (4.92 g, 95% yield).

1H NMR (400 MHz, CDCl₃) delta ppm 7.77 (m, 1H), 7.71 (m, 1H), 7.64 (m,1H), 3.93 (s, 3H).

Step b) Methyl 4-acetoxy-3-fluorobenzoate

Allyl chloride (105 μL, 1.28 mmol) and TFA (20 μL, 0.26 mmol) were addedto a suspension of zinc dust (480 mg, 7.34 mmol) and anhydrouscobalt(II) bromide (96.6 mg, 0.44 mmol) in MeCN (4 mL), under inert gas.After stirring at room temperature for 10 min, the aryl bromide (1.003g, 4.30 mmol dissolved in 5 mL MeCN) from (a) was added, followed byacetic anhydride (0.45 mL, 4.79 mmol) and more MeCN (1 mL). The mixturewas stirred overnight, quenched with 1M HCl (20 mL), and then extractedwith EtOAc (3×20 mL). The organic phase was washed successively withsaturated aqueous NaHCO₃ (20 mL) and saturated NaCl (2×20 mL), dried(Na₂SO₄), and concentrated. Flash chromatography (silica, 6/1 to 4/1petroleum ether-EtOAc gave recovered bromide (161.1 mg, 16%) and thedesired ketone (white solids, 305.5 mg, 36%).

NMR (CDCl₃) δ ppm: ¹H (400 MHz) 7.94-7.86 (m, 2H), 7.80 (dd, 1H, J=11.2,1.6 Hz), 3.95 (s, 3H), 2.67 (d, 3H, J=4.4 Hz); ¹⁹F (376 MHz) −109.2 (m);¹³C (100 MHz) 195.4 (d, J=3.7 Hz), 165.1 (d, J=2.2 Hz), 161.6 (d, J=255Hz), 135.8 (d, J=8.1 Hz), 130.7 (d, J=2.9 Hz), 129.0 (d, J=14 Hz), 125.2(d, J=3.6 Hz), 117.9 (d, J=26 Hz), 52.7 (s), 31.4 (d, J=7.3 Hz).

Step c) Methyl 4-(2-bromoacetoxy)-3-fluorobenzoate

THF (10 mL) and 2-pyrrolidinone (91 μL, 1.20 mmol) were added to amixture of the ketone from b) (198 mg, 1.01 mmol) and pyrrolidonehydrotribromide (532 mg, 1.07 mmol). After heating at 60-65° C. for 2 h,the mixture was concentrated under vacuum and then partitioned betweenEtOAc (20 mL) and saturated Na₂S₂O₃ (10 mL). The aqueous phase wasextracted with EtOAc (10 mL). The organic phases were combined, washedwith saturated NaCl (2×10 mL), dried (Na₂SO₄), and concentrated. Flashchromatography (silica, 7/1 petroleum ether-EtOAc) gave white solids(0.2634 g) containing 84% of the desired bromide (as determined byintegration of ¹⁹F NMR peaks).

NMR (CDCl₃) δ ppm: ¹H (400 MHz) 7.93 (m, 1H), 7.88 (m, 1H), 7.79 (dd,1H, J=11.2, 1.6 Hz), 4.50 (d, 2H, J=2.4 Hz), 3.94 (s, 3H); ¹⁹F (376 MHz)−108.4 (m).

Step d) Methyl3-fluoro-4-[2-(4-methylpiperazin-1-yl)-thiazol-4-yl]benzoate

EtOH (5.0 mL) was added to the bromoketone above (193 mg, 0.70 mmol) and4-methyl-piperazine-1-carbothioic acid amide (113 mg, 0.71 mmol) and themixture was heated at 70° C. for 2 h 15 min. The precipitates werefiltered, washed with cold EtOH, and dried under vacuum andcharacterized. The procedure was repeated in a larger scale for 1.75 gbromoketone (6.36 mmol).

NMR (1/1 CDCl₃-CD₃OD) δ ppm: ¹H (400 MHz) 8.20 (m, 1H), 7.86 (dd, 1H,J=8.4, 1.6 Hz), 7.76 (dd, 1H, J=11.4, 1.8 Hz), 7.38 (d, 1H, J=2.4 Hz),4.23 (br, 2H), 3.95, (s, 3H), 3.65 (br, 4H), 3.32 (br, 2H), 2.98 (s,3H); ¹⁹F (376 MHz) −114.0 (m). LCMS [M+H]+=336.

The precipitates from both preparations were combined and suspended insaturated NaHCO₃ (50 mL). The mixture was extracted with EtOAc. Theorganic phase was washed with water, dried (Na₂SO₄), and evaporated togive the title compound as cream solids (1.76 g).

Step e) 3-fluoro-4-[2-(4-methylpiperazin-1-yl)-thiazol-4-yl]benzoic acidHCl salt

The methyl ester (1.76 g, 5.25 mmol) from (d) was heated at 80° C. with6M HCl (40 mL) for 5.5 h. More 6M HCl (10 mL) was added and the mixturewas heated at 90° C. for 1 h 15 min. After cooling, the mixture was thenevaporated under vacuum and freeze-dried from water to give the finalproduct as cream solids in quantitative yield.

NMR (DMSO-d6) δ ppm: ¹H (400 MHz) 11.60 (br, 1H), 8.18 (t, 1H, J=8.0Hz), 7.82 (dd, 1H, J=8.4, 1.6 Hz), 7.72 (dd, 1H, J=12.0, 1.6 Hz), 7.48(d, 1H, J=2.8 Hz), 4.11 (m, 2H), 3.58 (m, 2H), 3.49 (m, 2H), 3.19 (m,2H), 2.80 (d, 3H, J=4.4 Hz); ¹⁹F (376 MHz) −113.5 (m); ¹³C (100 MHz)168.9, 166.0, 159.0 (d, J=250 Hz), 143.4, 131.4 (d, J=8 Hz), 129.8,125.8 (d, J=11 Hz), 125.6, 116.6 (d, J=24 Hz), 111.1 (J=15 Hz), 51.1,45.0, 41.9. LCMS [M+H]+=322.

Reference Example 3 6-aldehyde-intermediate

6-Formyl-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acidtert-butyl ester Step a

(3 as, 6aS)-6R-benzyloxy-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carboxylicacid benzyl ester (1a)

Dess-Martin reagent (12.5 g, 30 mmol) was dissolved in DCM (250 mL).6-Benzyloxy-3-hydroxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acidbenzyl ester (prepared as described in WO05/066180) (7.4 g, 20 mmol) inDCM (50 mL) was added to a stirred solution of oxidant at rt under anitrogen atmosphere over 45 min. After an additional 90 min stirring thereaction was deemed to be complete by TLC. Aqueous 10% Na₂S₂O₃ (200 mL)was added and the mixture was stirred at rt for another 15 minutes. Thetwo phase system was transferred into a separation funnel and extractedtwice with EtOAc (200 mL and 100 mL respectively). The combined organicphases were washed once with aqueous saturated NaHCO₃ (100 mL) and brine(100 mL), dried over Na₂SO₄, filtered and the solvent removed in vacuo,yielding the crude product title compound as a clear oil (7.69 g); ESI+,m/z: 368 (M+ +1).

Step b

(3aS,6aS)-6R-benzyloxy-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylicacid benzyl ester (1b)

The keto derivative (1a) (7.6 g) was dissolved in dry methanol (100 mL).Trimethyl orthoformate (30 mL) and pTsOH (0.2 g) was added at rt under anitrogen atmosphere. The mixture was heated at 60° C. for 8 hours. Whenthe reaction was deemed to have reached completion according to TLC, itwas cooled to rt and concentrated in vacuo. The crude product waspurified by column chromatography over silica gel eluting with ethylacetate-heptane (1:4) which gave the title compound as a clear oil (5.9g, 71% over 2 steps); ESI+, m/z: 382 (M+ −OMe).

Step c

(3aS,6aS)-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrol-6R-ol (1c)

A solution of compound (1b) (2.5 g, 6.4 mmol) in methanol (60 mL) andPd(OH)₂ (0.7 g) was stirred at rt under H₂ atmosphere for 48 hours. Whenthe reaction was deemed to have reached completion according to TLC, themixture was filtered and concentrated in vacuo to yield the crude titlecompound as a brownish oil (1.15 g); ESI+, m/z: 190 (M+ +1).

Step d

(3aS,6aS)-6R-hydroxy-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylicacid tert-butyl ester (1d)

3,3-Dimethoxy-hexahydro-furo[3,2-b]pyrrol-6-ol (1c) (2.80 g, 14.8 mmol)was dissolved in 75 mL of a mixture of dioxan/water (2:1). A solution of10% Na₂CO₃ (25 mL) was added drop wise to pH 9-9.5. The mixture wascooled to 0° C. in an ice-water bath and Boc anhydride was added in oneportion. The reaction was stirred at rt overnight and the pH of themixture was maintained at 9-9.5 by addition of more 10% solution ofNa₂CO₃ if necessary. The reaction was monitored by TLC (50:50 ethylacetate:isohexane). Once completed, the mixture was filtered toeliminate the salts formed and the solvent was evaporated in vacuo. Theaqueous mixture was extracted with 3×100 mL EtOAc, the combined organicphases were washed with 100 mL of water and 100 mL brine, dried overNa₂SO₄, filtered and the solvent was evaporated in vacuo to afford 3.79g of the title carbamate as a clear oil (89%), 94% pure (HPLC), ESI⁺,m/z: 312 (M⁺+Na).

Step e

3,3-Dimethoxy-6-oxo-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acidtert-butyl ester (1e)

To the alcohol (1e) (3.674 g, 12.70 mmol) dissolved in DCM (80 mL) wasadded Dess-Martin Periodinane (7.00 g, 16.5 mmol) and the solution wasstirred for 3 h at room temperature. The reaction was then quenched bythe addition of 10% Na₂S₂O₃ (aq) (150 mL) and the resulting slurry wasstirred for 15 minutes. The mixture was transferred to a separationfunnel and the phases were separated. The aqueous phase was extractedtrice with DCM and the combined organic phases were subsequently washedtwice with sat. NaHCO₃ solution and were the dried, filtered, andconcentrated. The crude material was purified by flash columnchromatography (toluene/ethyl acetate 3:1) which gave the title compound(2.882 g, 79%).

Step f

3,3-Dimethoxy-6-methylene-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acidtert-butyl ester (1f)

The keto compound 1e (1.10 g, 3.83 mmol) was dissolved in dry THF (30mL) and the solution was cooled to 0° C. A solution of methyltriphenylphosphonium bromide (4.0 g, 11.2 mmol) and KOtBu (1.17 g, 10.5mmol) in dry THF (40 mL) was added in 3 aliquots with 2 hours interval.After 6 hrs the solution was poured into a separatory funnel withdiethyl ether (70 mL) and extracted with 10% citric acid _((aq))(2*40mL). The organic phase was washed with saturated aqueous NaHCO₃ (40 mL),dried with Na₂SO₄, filtered and the solvent was evaporated in vacuo. Thecrude product was purified by flash chromatography (heptane: ethylacetate 4:1) which gave the title compound (524 mg, 48%)

¹H NMR (CDCl₃, 400 MHz) δ 1.48 (s, 9H), 3.27 (s, 3H), 3.40 (d, 3H,J=16.6), 3.57-3.64 (m, 1H), 3.84 (d, 1H, J=9.5), 3.92 (d, 1H, J=16.3),4.07-4.25 (m, 1H), 4.35-4.49 (m, 1H), 4.98 (bs, 1H), 5.22 (d, 1H,J=16.4), 5.34 (s, 1H).

Step g

6-Hydroxymethyl-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylicacid tert-butyl ester (1g)

The olefin 1e (524 mg, 1.84 mmol) was dissolved in dry THF (70 mL).9-BBN—H (0.5 M in THF) (7.34 mL, 3.67 mmol) was added and the solutionwas stirred over night. The solvent was removed by rotary evaporationand redissolved in THF (20 mL). MeOH (10 mL) was slowly added to thesolution and when the gas evolution had ceased, H₂O (20 mL) was added tothe solution followed by NaBO₃. The solution was filtered after it hadbeen stirred for 18 hrs and the filtrate was diluted with EtOAc (70 mL)and washed with brine (2*50 mL). The organic phase was dried withNa₂SO₄, filtered and the solvent was evaporated in vacuo. The crudeproduct was purified by flash chromatography (heptane: ethyl acetate2:1) which gave the title compound (477 mg, 86%).

¹H NMR (CDCl₃, 400 MHz) δ1.47 (s, 9H), 2.09-2.25 (m, 2H), 3.02-3.20 (m,1H), 3.29 (s, 3H), 3.39 (s, 3H), 3.65-3.93 (m, 4H), 4.44 (d, 1H, J=5.7),4.70-4.84 (m, 1H).

Step h

6-Formyl-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acidtert-butyl ester (1h)

To a solution of the alcohol 1g (370 mg, 1.22 mmol) dissolved in dry DCM(10 mL) was added Dess Martin periodinane (673 mg, 1.59 mmol). Thereaction was stirred for 40 minutes and then quenched by addition of 10mL of 10% Na₂S₂O₃: NaHCO_(3(sat)) 1:1. The solution was diluted with DCM(50 mL) and extracted with a 1:1 mixture of 10% Na₂S₂O₃: NaHCO_(3(sat))(50 mL). The organic phase was dried with Na₂SO₄, filtered andevaporated. The crude product was purified by flash chromatography(heptane: ethyl acetate (2:1) which gave the title compound (290 mg,79%).

¹H NMR (CDCl₃, 400 MHz) δ1.47 (s, 9H), 2.90-3.06 (m, 1H), 3.29 (s, 3H),3.38 (s, 3H), 3.67-3.85 (m, 2H), 3.88-4.55 (m, 3H), 4.93-5.19 (m, 1H),9.64* and 9.80* (s, 1H). * Two peaks due to rotamers.

Reference Example 4 6-Acetylene P1 Building Block

6-Ethynyl-3,3-dimethoxy-hexahydro-furo(3,2-b)pyrrole-4-carboxylic acidtert butyl ester Step a

The aldehyde of Reference Example 3 (318.3 mg, 1.06 mmol) in 6 mLdichloromethane (DCM) was added dropwise to a solution of CBr₄ (700 mg,2.11 mmol) and PPh₃ (1.10 g, 4.19 mmol) in 10 mL DCM, with cooling in anice bath. After stirring at 0° C. for 2 h, the mixture was diluted with30 mL iso-hexane and then filtered through a short Celite column. Thecolumn was washed with 20 mL i-hexane, followed by 3/1 i-hexane-DCM. Thefiltrate was evaporated to give light yellow solids. Flashchromatography (silica, 3/1 i-hexane-EtOAc) gave compound 2 (339.5 mg,70% yield).

1H NMR (400 MHz, CDCl₃) δ 6.55 (d, 1H, J=8.8 Hz, HC═CBr₂), 4.64 (br s,1H), 4.42 (d, 1H, J=5.2 Hz), 4.10-3.90 (br m, 1H), 3.85-3.65 (m, 2H),3.37 (s, 3H, OMe), 3.29 (s, 3H, OMe), 3.00 (m, 1H), 2.84 (br s, 1H).

Step b

Butyllithium solution (1.6M in hexanes, 1.50 mL) was added dropwise at−78° C. to a solution of the dibromoalkene of step a (327.5 mg, 0.72mmol) in 13 mL THF. After stirring at −78° C. for 1.75 h, the reactionwas quenched with 3 mL saturated aqueous NH₄Cl. The reaction mixture wasconcentrated, then partitioned between 30 mL saturated aqueous NaCl and30 mL EtOAc. The aqueous phase was extracted with 2×30 mL EtOAc. Theorganic phases were combined, dried (Na₂SO₄), and evaporated to give ayellow oil. Flash chromatography (silica, 3/1 i-hexane-EtOAc) gave titlecompound as white solids (161.7 mg, 67% yield).

1H NMR (500 MHz, CDCl₃) δ 4.67 (br s, 1H), 4.40 (br s, 1H), 4.16-4.0 (brm, 1H), 3.88-3.74 (m, 2H), 3.37 (s, 3H, OMe), 3.29 (s, 3H, OMe), 3.16(br s, 1H), 2.81 (br s, 1H, HC—C≡CH), 2.20 (d, 1H, C≡CH), 1.47 (s, 9H,tBu).

Reference Example 5 A Typical P1/P2 Coupling & Deprotection Step a

Acetyl chloride (0.51 mL) was added dropwise to a solution of theterminal alkyne of Reference Example 4 (153 mg, 0.514 mmol) in MeOH (4.6mL), chilled in an ice bath. The reaction mixture was stirred at RTovernight and then evaporated. Boc-Leu-OH—H₂O (145 mg, 0.58 mmol) wasadded and the mixture was coevaporated from DMF, then redissolved in 5mL DMF, and cooled in an ice bath. DIEA (0.36 mL, 2.1 mmol) was added,followed by HATU (220 mg, 0.578 mmol). After stirring at 0° C. for 15min, the mixture was stirred at RT for 3 h and then concentrated. Themixture was dissolved in 20 mL EtOAc, washed successively with saturatedaqueous NaHCO₃ (10 mL) and saturated aqueous NaCl (2×10 mL), dried(Na₂SO₄), and evaporated to give a yellow-brown oil. Flashchromatography (silica, 1/1 i-hexane-EtOAc) gave title compound (213 mg,quantitative).

HPLC-MS: single peak, mass 411 [M+H]⁺, R_(t)=3.15 min (gradient 5 to 99%B in 3 min, then 100% B for 1.5 min)

Method—Flow: 0.8 mL/min, UV=210-400 nm, ACE C8 3×50 mm; Mobile phase A:10 mM NH₄Ac in 90% H₂O, B: 10 mM NH₄Ac in 90% MeCN

Step b

Boc deprotection of the compound of step a) (0.514 mmol) was done as forReference Example 4 above to give the title HCl salt. The salt wasdissolved in CH₂Cl₂ and divided into three portions, evaporatedseparately to give 0.17 mmol per sample.

Reference Example 6 An Alternative P3 Building Block

Availability of Starting Materials—

Methyl 4-acetylbenzoate is available from Aldrich;4-methyl-piperazine-1-carbothioic acid amide—11 suppliers found inSciFinder (perhaps Chem Pur Products Ltd in Germany most convienient).

Step a) 4-(2-Bromo-acetyl)-benzoic acid methyl ester

To a solution of 4-acetyl-benzoic acid methyl ester (8.4 mmol) in aceticacid (20 mL) was added bromine (8.4 mmol). The reaction was stirred atRT for 2 h over which time the red colour disappeared and an off whiteprecipitate formed. The product was collected by filtration and washedwith cold methanol/water (200 mL 1:1) to yield a white powder (55%). 1HNMR (400 MHz, CDCl₃) 3.98 (3H, s), 4.20 (2H, s), 8.02 (2H, d, J=8 Hz),8.18 (2H, d, J=8 Hz).

Step b) 4-(2-Fluoro-acetyl)-benzoic acid methyl ester

To a suspension of potassium fluoride (3.11 mmol) in acetonitrile (1 mL)was added 18-crown-6 (0.1 mmol) and the reaction was heated at 90° C.for 30 mins. 4-(2-Bromo-acetyl)-benzoic acid (1.56 mmol) was added andthe reaction heated at 90° C. for 16 h. The reaction was diluted withwater (10 mL) and extracted with ethyl acetate (3×20 mL). The productwas purified on silica eluting with 5-15% ethyl acetate in iso-hexane toyield on concentration in vacuo of the desired fractions, the titleproduct as a white solid (31%). ¹H NMR (400 MHz, CDCl₃) 3.98 (3H, s),5.55 (2H, d, J=50 Hz), 7.95 (2H, d, J=8 Hz), 8.18 (2H, d, J=8 Hz).

Step c) 4-(2-Bromo-2-fluoro-acetyl)-benzoic acid methyl ester

To a suspension of 4-(2-fluoro-acetyl)-benzoic acid (1.19 mmol) inacetic acid (5 mL) was added bromine (1.19 mmol). The reaction washeated at 45° C. for 4 h over which time a green solution formed. Thereaction was concentrated in vacuo and azeotroped twice with toluene toyield the title compound as a green solid (100%). The product was usedcrude in the next step. 1H NMR (400 MHz, CDCl₃) 3.98 (3H, s), 7.04 (1H,s), 8.05-8.10 (4H, m).

Step d) 4-[5-Fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoicacid methyl ester

4-(2-Bromo-2-fluoro-acetyl)-benzoic acid methyl ester (1.18 mmol) and4-methyl-piperazine-1-carbothioic acid amide (1.18 mmol) were dissolvedin ethanol (10 mL). The reaction was heated at reflux for 2 h. Thereaction was cooled to RT causing the product to precipitate. Theproduct was collected by filtration and washed with cold ethanol. Theproduct was given an aqueous sodium bicarbonate work up to yield thetitle compound as a colourless oil (74%). MS (ES+) 337 (M+H, 100%).

Step f 4-[5-Fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoicacid di-hydrochloride

To a solution of4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acidmethyl ester (0.43 mmol) in tetrahydrofuran/water (2.5 mL, 4:1) wasadded lithium hydroxide (0.5 mmol). The reaction was stirred at RT for16 h. The reaction was concentrated in vacuo and hydrochloric acid (2N,3 mL) was added causing the product to precipitate as a white solid. Theproduct was collected by filtration to yield the title product as awhite solid (79%). MS (ES+) 322 (M+H, 100%).

Reference Example 7 An Alternative P1 Building Block

Step a) Oxidation of Alcohol 7-1

A solution of compound 7-1 (0.85 g, 2.8 mmol) (see reference example 3step g) in dry CH₂Cl₂ (15 mL) was purged with argon for 30 minutes.Dess-Martin periodinone (1.78 g, 4.2 mmol) was added at 0° C. and thereaction mixture was stirred at room temperature for 2 hours. When thereaction was deemed to have reached completion, the reaction wasquenched with 10% Na₂S₂O₃ solution. The reaction mixture was dilutedwith CH₂Cl₂ (30 mL), and then washed in turn with saturated Na₂S₂O₃solution (2×50 mL), and saturated sodium bicarbonate solution (2×50 mL).The organic layer was then dried over anhydrous sodium sulfate, filteredand the filtrate conc in vacuo at room temperature. The crude productaldehyde 7-2 (0.75 g, crude) was used in the next step without furtherpurification.

TLC: EtOAc:Pet ether 1:1 R_(f)=0.5.

Step b) Wittig Reaction with Compound 7-2

To a stirred solution of isopropyltriphenylphosphonium iodide (6.48 g,0.015 mol) (coevaporated with dry toluene prior to start of thereaction) in dry THF (20 mL) was added a solution of n-BuLi (10.7 mL,0.0172 mol, 1.6M in hexane) at −10° C. under nitrogen atmosphere andfurther stirred at 0° C. for 1 hour. The color of the reaction mixtureslowly turned to dark orange color. A solution of compound 2 (0.75 g,0.0025 mol) and anhydrous lithium chloride (˜1.5 mg) in dry THF (20 mL)was slowly added to the reaction mixture at 0° C. under a nitrogenatmosphere. The reaction mixture was further stirred for 30 minutesmaintaining the same temperature and then slowly warmed to rt andstirred for another 30 minutes. The reaction mixture was then quenchedwith saturated ammonium chloride solution (20 mL). The crude product wasextracted with EtOAc (2×30 mL) and the organic layer was washed withbrine (50 mL), and dried over anhydrous sodium sulfate. The solvent wasremoved in vacuo and the crude product was purified by columnchromatography (neutral alumina, eluent 1% EtOAc in pet ether) to affordpure compound 7.3 (0.19 g, yield 21% over two steps).

Reference Example 8 An Alternative P1−P2 Building Block

The initial reaction was carried out in a similar way as reported forreference example 5 but instead using 4 (0.2 g, 0.67 mmol) in dry MeOH(15 mL) and acetyl chloride (0.66 mL) to get the Boc-deprotected aminecompound which was then treated with Boc-cyclopentyl glycine (0.17 g,0.7 mmol), HATU (0.27 g, 0.7 mmol) and DIEA (0.45 mL, 2.6 mmol) in dryDMF (7 mL) to get the pure title compound [0.195 g, yield 68%].

TLC system: EtOAc:pet ether 1:1 v/v, R_(f)=0.4

Reference Example 9 Probe Compound for the Feasibility of Dihalovinyl P1

This compound tests the synthetic and biological feasibility of adihalovinyl P1 end product. It will be appreciated that introduction of6-difluorovinyl by the corresponding Wittig reaction of referenceexample 3 is trivial and that preparation of a biologically activeend-product provides confidence that the claimed difluorvinyl compoundsare also synthetically stable and active.

Step a

The aldehyde 9-1 (318.3 mg, 1.06 mmol) (see reference example 3) in 6 mLdichloromethane (DCM) was added dropwise to a solution of CBr₄ (700 mg,2.11 mmol) and Ph₃P (1.10 g, 4.19 mmol) in 10 mL DCM, with cooling in anice bath. After stirring at 0° C. for 2 h, the mixture was diluted with30 mL iso-hexane and then filtered through a short Celite column. Thecolumn was washed with 20 mL i-hexane, followed by 3/1 i-hexane-DCM. Thefiltrate was concentrated in vacuo to give light yellow solids. Flashchromatography (silica, 3/1 i-hexane-EtOAc) gave compound 9-2 (339.5 mg,70% yield).

1H NMR (400 MHz, CDCl₃) δ 6.55 (d, 1H, J=8.8 Hz, HC═CBr₂), 4.64 (br s,1H), 4.42 (d, 1H, J=5.2 Hz), 4.10-3.90 (br m, 1H), 3.85-3.65 (m, 2H),3.37 (s, 3H, OMe), 3.29 (s, 3H, OMe), 3.00 (m, 1H), 2.84 (br s, 1H).

Rf (TLC 1/1 isohexane-EtOAc) 0.79.

HPLC-MS: ion cluster [M+Na]⁺ 478 (8%), 480 (15%), 482 (7%), R_(t)=3.56min (gradient 5 to 99% B in 3 min, then 100% B for 1.5 min)

Step b

Acetyl chloride (0.18 mL) was added dropwise to a solution of thedibromoalkene 9-2 (81.7 mg, 0.179 mmol) in MeOH (1.62 mL), chilled in anice bath. The reaction mixture was stirred at RT overnight and thenevaporated. Boc-Leu-OH—H₂O (50 mg, 0.20 mmol), HATU (75 mg, 0.20 mmol),1.8 mL DMF, and lastly, DIEA (125 μL, 0.72 mmol) were added. Afterstirring at RT for 5.5 h the mixture was concentrated in vacuo, and thenpartitioned between EtOAc and saturated aqueous NaHCO₃. The organicphase was washed twice with saturated aqueous NaCl, dried (Na₂SO₄), andconcentrated in vacuo. Flash chromatography (silica, 3/1isohexane-EtOAc) gave compound 9-3 as a white solid (86.3 mg, 85%yield).

Rf (TLC 1/1 isohexane-EtOAc) 0.54.

HPLC-MS: mass 571 [M+H]⁺; R_(t)=3.75 min 96% (gradient 5 to 99% B in 3min, then 100% B for 1.5 min)

Method—Flow: 0.8 mL/min, UV=210-400 nm, ACE C8 3×50 mm; Mobile phase A:10 mM NH₄Ac in 90% H₂O, B: 10 mM NH₄Ac in 90% MeCN

Step c

Boc deprotection of compound 9-3 (86.3 mg, 0.15 mmol) was done as for9-2 above to give the amine HCl salt. DMF (1.5 mL) was added to amixture of the amine salt,4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acid hydrobromide (65mg, 0.17 mmol), and HATU (65 mg, 0.17 mmol) with cooling in an ice bath.DIEA (120 μL, 0.69 mmol) was added. After stirring at RT for 3 h, 20 mLEtOAc was added, and then the mixture was washed with 1M NaHCO₃ (10 mL),followed by saturated aqueous NaCl. The organic phase was dried (Na₂SO₄)and concentrated in vacuo to give crude product. Flash chromatography(silica, CH₂Cl₂-MeOH 100/1 to 100/4) gave the desired product as a paleyellow solid (65.7 mg, 58% yield).

HPLC-MS: mass 756 [M+H]⁺; R_(t)=3.83 min (5 to 99% B in 3 min, then 100%B for 1.5 min) and R_(t)=4.07 min (30 to 80% B in 3 min, then 100% B for1.5 min) 96% pure in two gradients

Step d

The ketal 9-4 (65.7 mg, 0.087 mmol) was stirred with 1.0 mL of 97.5/2.5(v/v) TFA-H₂O for 4 h 20 min and then quenched with an aqueoussuspension of NaHCO₃. The mixture was extracted with EtOAc. The organicphase was washed with saturated aqueous NaCl, dried (Na₂SO₄), andconcentrated in vacuo. Purification by prep HPLC gave the ketone 9-5 asa white solid (12.6 mg).

1H NMR (500 MHz, CDCl₃) 2 rotomers, major: δ 7.90 and 7.81 (ABq, 4H,phenyl), 6.91-6.87 (2H, thiazole and NH), 6.67 (d, 1H, HC═CBr₂), 5.05(td, 1H, Leu CHα), 4.85 (m, 1H, bicyclic bridge HCO), 4.74 (d, 1H,bicyclic bridge HCN), 4.32 and 3.42 (1H each, bicyclic NCH₂), 4.18 and4.00 (ABq, 2H, OCH₂), 3.59 (4H, piperazine CH ₂N-thiazole), 3.25 (1H,Br₂C═CH—CH), 2.56 (4H, piperazine CH ₂NMe), 2.37 (s, 3H, NMe), 1.80-1.54(d, 3H, CH ₂CHMe₂), 1.04 (d, 3H, CHMe ₂), 0.94 (d, 3H, CHMe ₂).

LC-UV/MS: monoisotopic molecular mass 709.1 Da, >94% purity

(Column: ACE C₈ 50×3.0 mm, 3 μm particles; Mobile phases A: 10 mM NH₄Ac,B: 10 mM NH₄Ac in 90% MeCN; gradient: 30-70% B in 10 min followed by awash for 2 min at 100% B; Flow: 0.8 mL/min, Detection: UV @ 210-400 nmand ESI-MS)

Example 1

N-[1-6-(ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamideStep a

DMF (2 mL) was added to a mixture of4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acid hydrobromide(73.9 mg, 0.19 mmol), the compound of Reference Example 5 (0.17 mmol),and HATU (73.4 mg, 0.19 mmol) with cooling in an ice bath. DIEA (0.12mL, 0.69 mmol) was added. After stirring at RT for 2.5 h, the mixturewas concentrated, redissolved in 20 mL EtOAc, and then washed with 10 mLsaturated aqueous NaHCO₃. The aqueous phase was extracted with 10 mLEtOAc. The organic phases were combined, washed with saturated aqueousNaCl (2×15 mL), dried (Na₂SO₄), and evaporated to give crude product.Initial flash chromatography (silica 40-63 μm, 5-8% MeOH in EtOAc) gavepurified material which was subjected to a second chromatography (YMCgel silica 6 nm S-50 μm, 1-5% MeOH in CH₂Cl₂) to give title compound aslight yellow solids (50.1 mg, 49% yield).

HPLC-MS: mass 596 [M+H]⁺, single peak, R_(t)=3.18 min (gradient 5 to 99%B in 3 min, then 100% B for 1.5 min)

Step b

The compound of step a) (50 mg, 0.0839 mmol) was dissolved in 10 mL ofTFA: H₂O (97.5:2.5) and stirred for 4 hours. The solvent was pored intoa separatory funnel, extracted with EtOAc and washed with saturatedaqueous NaHCO₃. The organic phase was dried with Na₂SO₄, filtered andevaporated. The crude product was purified by semi-prep. HPLC on aXBrideg Phenyl 5 μm column with mobile phases A (90:10 H₂O:acetonitrile, 10 mM NH₄Ac) and B (10:90 H₂O: acetonitrile, 10 mM NH₄Ac)going from 25-60% B. The product was obtained as a white solid in 62%yield (29 mg). LRMS (M+H) 550.

¹H NMR (CDCl₃, 400 MHz): 0.95 (d, J=6.4, 3H), 1.03 (d, J=6.4, 3H),1.57-2.10 (m 4H), 2.37 (s, 3H), 2.60-2.54 (m, 4H), 3.17-3.26 (m, 1H),3.55-3.64 (m, 5H), 4.07 (d, J=17.1, 1H), 4.27 (d, J=17.2, 1H), 4.50 (dd,J=7.8, 9.9, 1H), 4.77 (d, J=5.1, 1H), 4.91 (dd, J=4.6, 4.6, 1H),4.97-5.06 (m, 1H), 6.85-6.91 (m, 2H), 7.79 (d, J=8.3, 2H), 7.89 (d,J=8.4, 3H).

Example 2

N-[1-6-(ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamideStep a

DMF (1.7 mL) was added to a mixture of4-[5-fluoro-2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acidhydrochloride (68.0 mg, 0.19 mmol), the compound of Reference Example 5(0.17 mmol), and HATU (73.8 mg, 0.19 mmol) with cooling in an ice bath.DIEA (0.12 mL, 0.69 mmol) was added. After stirring at RT for 2.75 h,the mixture was treated as for Example 1 to give the crudefluorothiazole analogue. Flash chromatography (YMC gel silica, 1-3% MeOHin CH₂Cl₂) gave the title compound as light yellow solids (62.8 mg, 60%yield).

HPLC-MS: mass 614 [M+H]⁺, single peak on UV, R_(t)=3.39 min (gradient 5to 99% B in 3 min, then 100% B for 1.5 min)

Step b

The compound of step a) (63 mg, 0.102 mmol) was dissolved in 10 mL ofTFA:H₂O (97.5:2.5) and stirred for 4 hours. The solvent was pored into aseparatory funnel, extracted with EtOAc and washed with sat.NaHCO_(3(aq)). The organic phase was dried with Na₂SO₄, filtered andevaporated. The crude product was purified by semi-prep. HPLC on aXBrideg Phenyl 5 um column with mobile phases A (90:10 H₂O:acetonitrile, 10 mM NH₄Ac) and B (10:90 H₂O: acetonitrile, 10 mM NH₄Ac)going from 25-60% B. The product was obtained as a white solid in 46%yield (26 mg). LRMS (M+H) 568.

¹H NMR (CDCl₃, 400 MHz): 0.95 (d, J=6.4, 3H), 1.03 (d, J=6.4, 3H),1.58-2.15 (m 4H), 2.37 (s, 3H), 2.61-2.48 (m, 4H), 3.18-3.25 (m, 1H),3.53-3.42 (m, 4H), 3.60 (t, J=10.5, 1H), 4.07 (d, J=17.1, 1H), 4.27 (d,J=17.2, 1H), 4.56-4.46 (m, 1H), 4.91 (t, J=4.5, 1H), 4.96-5.06 (m, 1H),6.87 (d, J=8.2, 1H), 7.81 (d, J=8.4, 2H), 7.90 (t, J=9.4, 2H).

Example 3

N-[1-6-(ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamideStep a

DMF (1.7 mL) was added to a mixture of3-fluoro-4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acidhydrochloride (68.9 mg, 0.19 mmol), the compound of Reference Example 5(0.17 mmol), and HATU (80 mg, 0.21 mmol) with cooling in an ice bath.DIEA (0.12 mL, 0.69 mmol) was added. After stirring at RT for 3 h, themixture was concentrated, redissolved in 30 mL EtOAc, washedsuccessively with 15 mL saturated aqueous NaHCO₃ and then 30 mLsaturated aqueous NaCl. The organic phase was dried (Na₂SO₄) and thenevaporated. Flash chromatography (YMC gel silica, 1-3% MeOH in CH₂Cl₂)gave the title compound as off-white solids (72.4 mg, 70% yield).

HPLC-MS: mass 614 [M+H]⁺, R_(t)=3.46 min (gradient 5 to 99% B in 3 min,then 100% B for 1.5 min

Step b

The ketal of step a) (67 mg, 0.11 mmol) was stirred with 1.10 mL of97.5/2.5 (v/v) TFA-H₂O for 2 h and then concentrated. The mixture wasdiluted with EtOAc (10 mL), washed with saturated aqueous NaHCO₃ (5 mL),followed by saturated aqueous NaCl (2×5 mL), dried (Na₂SO₄), andevaporated. The crude material was dissolved in 2 ml MeCN and 1 mL H₂O,and 0.9 ml of this solution was purified by prep HPLC to give the ketoneof the title I compound as white solids (8.2 mg).

1H NMR (500 MHz, CDCl₃) 2 rotomers δ 8.22 (m, 1H, Ph), 7.63-7.55 (m, 2H,Ph), 7.21 (m, 1H, thiazol), major 6.91 and minor 6.87 (d, 1H, J=8.0 and7.5 Hz, NHC═O), minor 5.05 and major 5.00 (m, 1H), 4.93 (m, 1H), 4.79(d, 1H, J=5.0 Hz), 4.48 (dd, 1H, J=10.2, 7.7 Hz), 4.28 and 4.09 (ABq, 1Heach), 3.64-3.59 (m, 5H), 3.23 (m, 1H, HC—C≡CH), 2.58 (m, 4H), 2.38 (s,3H, NMe), 2.35 (d, 1H, J=2.0 Hz, C≡CH), 1.78-1.59 (m, 3H), 1.04 (d, 3H,J=6.0 Hz), 0.96 (d, 3H, J=6.5 Hz).

LC-UV/MS: monoisotopic molecular mass 567.2 Da, 97.6% purity

(Column: ACE C8 50×3.0 mm, 3 μm particles; Mobile phases A: 10 mM NH₄Ac,B: 10 mM NH₄Ac in 90% MeCN; gradient: 20-100% B in 10 min followed by awash for 2 min at 100%; Flow: 0.8 mL/min, Detection: UV @ 210-400 nm andESI-MS)

Example 4N-[1-6-(dimethylvinyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamide

Acetyl chloride (0.3 mL) was added dropwise to an ice-cooled solution ofcompound 7-3 (103.4 mg, 0.316 mmol) (see reference example 7) inmethanol (2.9 mL). The solution was stirred at rt overnight, and thenconcentrated in vacuo, coevaporated twice with CH₂Cl₂, and dried undervacuum. Boc-L-leucine-H₂O (96.2 mg, 0.386 mmol) and HATU (143 mg, 0.376mmol) were added and the mixture was cooled in an ice bath. DMF (3.2mL), followed by DIEA (220 μL, 1.26 mmol) were added. The resultingsolution was stirred at rt for 6 h. The reaction mixture wasconcentrated in vacuo, redissolved in EtOAc (15 mL), and washedsuccessively with saturated aqueous NaHCO₃ (10 mL) and saturated aqueousNaCl (10 mL). The organic phase was dried (Na₂SO₄) and then concentratedin vacuo to give the crude material. Flash column chromatography(silica, 2/1 isohexane-EtOAc) gave compound 4i as white solids (127.8mg, 92% yield).

TLC R_(f)=0.56 (1/1 isohexane-EtOAc)

LC-UV/MS R_(t)=1.98 min (single peak), mass 441 [M+H]⁺ (gradient 70 to99% B in 3 min, then 100% B for 1.5 min (Method—Flow 0.8 mL/min,UV=210-400 nm, Phenomenex Gemini-NX 3 μm C18 110 Å 50×3.0 mm, Mobilephases A: 10 mM NH₄Ac in H₂O, B: 10 mM NH₄Ac in 90/10 MeCN—H₂O)

Compound 4-i (127.8 mg, 0.290 mmol) was deprotected as above using MeOH(2.7 mL) and acetyl chloride (0.30 mL). The coupling of the resultingamine HCl salt (half of material, 0.145 mmol) with4-[5-fluoro-2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acidhydrochloride (67.4 mg, 0.19 mmol) was done similarly as the Boc-Leu-OHcoupling step, in DMF (2.0 mL) with HATU (69 mg, 0.18 mmol) and DIEA(115 μL, 0.66 mmol) for 3.5 h. After flash column chromatography of thecrude product (silica, 2-5% MeOH in CH₂Cl₂), compound 4-ii was obtainedas pale yellow solids (48.3 mg, 52% yield).

TLC Rf=0.5 (9/1 CH₂Cl₂—MeOH)

LC-UV/MS Rt=2.41 min, 95q % pure, mass 644 [m+H]⁺ (gradient 70 to 99% Bin 3 min, then 100% B for 1.5 min

To an ice-cooled solution of the ketal 4-ii (41.3 mg, 0.064 mmol) inCH₂Cl₂ (0.3 mL) was added dropwise 0.65 ml of a solution of TFA-water(97.5/2.5 v/v). The reaction mixture was stirred for 30 min at rt, andthen replaced in an ice bath and quenched with saturated aqueous NaHCO₃(10 mL). The mixture was extracted twice with EtOAc (20 mL, 10 mL). Theorganic phases were combined, washed with saturated aqueous NaCl (10mL), dried (Na₂SO₄), and concentrated in vacuo. Purification by prepHPLC gave the final title compound.

LC-UV/MS Rt=6.5 and 7.5 min (hydrate and ketone), 98% pure, monoisotopicmolecular mass 597.3 Da (Method—Flow 0.8 mL/min; UV=210-400 nm andESI-MS; Phenomenex Gemini-NX C18 50×3.0 mm, 3 μm particles; Mobilephases A: 5 mM NH₄Ac in H₂O, B: 5 mM NH₄Ac in MeCN, gradient 20-99% B in10 min followed by a wash for 2 min at 100% B)

Example 5N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamideStep a

The reaction was carried out in a similar way as reported in case ofExample 3 but using the P1−P2 building block of reference example 8(0.14 g, 0.33 mmol) in dry MeOH (15 mL) and acetyl chloride (0.5 mL) wasused to get the Boc-deprotected amine which was further treated with theHCl salt (0.132 g, 0.33 mmol), HOBt (0.04 g, 0.3 mmol), EDC.HCl (0.117g, 0.61 mmol) and NMM (0.11 mL, 1.0 mmol) in dry DMF (10 mL) to get thepure compound 5-a [0.05 g, yield 26%].

TLC system: CHCl₃:MeOH 9.5:0.5 v/v, R_(f)=0.25.

Step b

Deprotection of ketal 5-a (40.5 mg, 0.065 mmol) to the title ketone wasconducted as for compound 3 using 0.65 mL of the TFA-water solution,with reaction at rt for 2 h 15 min. Purification by prep HPLC gave 11 aspale yellow solids (14.75 mg).

LC-UV/MS Rt=5.3 and 6.0 min, (hydrate and ketone), 96.7% pure,monoisotopic molecular mass 579.2 Da (Method—Flow 0.8 mL/min; UV=210-400nm and ESI-MS; Phenomenex Gemini-NX C18 50×3.0 mm, 3 μm particles;Mobile phases A: 5 mM NH₄Ac, B: 5 mM NH₄Ac in MeCN, gradient 20-99% B in10 min followed by a wash for 2 min at 100% B)

BIOLOGICAL EXAMPLES Determination of Cathepsin K Proteolytic CatalyticActivity

Convenient assays for cathepsin K are carried out using humanrecombinant enzyme, such as that described in PDB.

ID BC016058 standard; mRNA; HUM; 1699 BP.

DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNA cloneMGC:23107

RX MEDLINE; RX PUBMED; 12477932.

DR RZPD; IRALp962G1234.

DR SWISS-PROT; P43235;

The recombinant cathepsin K can be expressed in a variety ofcommercially available expression systems including E coli, Pichia andBaculovirus systems. The purified enzyme is activated by removal of theprosequence by conventional methods.

Standard assay conditions for the determination of kinetic constantsused a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, andwere determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTAand 10 mM 2-mercaptoethanol or 100 mMNa phosphate, imM EDTA, 0.1%PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20mM cysteine, in each case optionally with 1M DTT as stabiliser. Theenzyme concentration used was 5 nM. The stock substrate solution wasprepared at 10 mM in DMSO. Screens were carried out at a fixed substrateconcentration of 60 μM and detailed kinetic studies with doublingdilutions of substrate from 250 μM. The total DMSO concentration in theassay was kept below 3%. All assays were conducted at ambienttemperature. Product fluorescence (excitation at 390 nm, emission at 460nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent platereader. Product progress curves were generated over 15 minutes followinggeneration of AMC product.

Cathepsin S Ki Determination

The assay uses baculovirus-expressed human cathepsin S and theboc-Val-Leu-Lys-AMC fluorescent substrate available from Bachem in a 384well plate format, in which 7 test compounds can be tested in parallelwith a positive control comprising a known cathepsin S inhibitorcomparator.

Substrate Dilutions

280 μl/well of 12.5% DMSO are added to rows B—H of two columns of a 96deep well polypropylene plate. 70 μl/well of substrate is added to rowA. 2×250 μl/well of assay buffer (100 mM Na phosphate, 100 mM NaCl, pH6.5) is added to row A, mixed, and double diluted down the plate to rowH.

Inhibitor Dilutions

100 μl/well of assay buffer is added to columns 2-5 and 7-12 of 4 rowsof a 96 well V bottom polypropylene plate. 200 μl/well of assay bufferis added to columns 1 and 6.

The first test compound prepared in DMSO is added to column 1 of the toprow, typically at a volume to provide between 10 and 30 times theinitially determined rough K_(i). The rough Ki is calculated from apreliminary run in which 10 μl/well of 1 mM boc-VLK-AMC (1/10 dilutionof 10 mM stock in DMSO diluted into assay buffer) is dispensed to rows Bto H and 20 μl/well to row A of a 96 well Microfluor™ plate. 2 μl ofeach 10 mM test compound is added to a separate well on row A, columns1-10. Add 90 μl assay buffer containing 1 mM DTT and 2 nM cathepsin S toeach well of rows B—H and 180 μl to row A. Mix row A using amultichannel pipette and double dilute to row G. Mix row H and read inthe fluorescent spectrophotometer. The readings are Prism data fitted tothe competitive inhibition equation, setting S=100 μM and K_(M)=100 μMto obtain an estimate of the K_(i), up to a maximum of 100 μM.

The second test compound is added to column 6 of the top row, the thirdto column 1 of the second row etc. Add 1 μl of comparator to column 6 ofthe bottom row. Mix column 1 and double dilute to column 5. Mix column 6and double dilute to column 10.

Using an 8-channel multistepping pipette set to 5×10 μl, distribute 10μl/well of substrate to the 384 well assay plate. Distribute the firstcolumn of the substrate dilution plate to all columns of the assay platestarting at row A. The tip spacing of the multichannel pipette willcorrectly skip alternate rows. Distribute the second column to allcolumns starting at row B.

Using a 12-channel multistepping pipette set to 4×10 μl, distribute 10μl/well of inhibitor to the 384 well assay plate. Distribute the firstrow of the inhibitor dilution plate to alternate rows of the assay platestarting at A1. The tip spacing of the multichannel pipette willcorrectly skip alternate columns. Similarly, distribute the second,third and fourth rows to alternate rows and columns starting at A2, B1and B2 respectively.

Mix 20 ml assay buffer and 20 μl 1M DTT. Add sufficient cathepsin S togive 2 nM final concentration.

Using the a distributor such as a Multidrop 384, add 30 μl/well to allwells of the assay plate and read in fluorescent spectrophotomoter suchas an Ascent.

Fluorescent readings, (excitation and emission wavelengths 390 nm and460 nm respectively, set using bandpass filters) reflecting the extentof enzyme cleavage of the fluorescent substrate, notwithstanding theinhibitor, are linear rate fitted for each well.

Fitted rates for all wells for each inhibitor are fitted to thecompetitive inhibition equation using SigmaPlot 2000 to determine V, Kmand Ki values.

Cathepsin L Ki

The procedure above with the following amendments is used for thedetermination of Ki for cathepsin L.

The enzyme is commercially available human cathepsin L (for exampleCalbiochem). The substrate is H-D-Val-Leu-Lys-AMC available from Bahcem.The assay buffer is 100 mM sodium acetate 1 mM EDTA, pH5.5) The DMSOstock (10 mM in 100% DMSO) is diluted to 10% in assay buffer. Enzyme isprepared at 5 nM concentration in assay buffer plus 1 mM dithiothreitoljust before use. 2 ul of 10 mM inhibitor made up in 100% DMSO isdispensed into row A. 10 μl of 50 μM substrate (=1/200 dilution of 10 mMstock in DMSO, diluted in assay buffer)

Inhibition Studies

Potential inhibitors are screened using the above assay with variableconcentrations of test compound. Reactions were initiated by addition ofenzyme to buffered solutions of substrate and inhibitor. K_(i) valueswere calculated according to equation 1.

$\begin{matrix}{v_{0} = \frac{VS}{{K_{M}\left( {1 + \frac{I}{K_{i}}} \right)} + S}} & (1)\end{matrix}$where v₀ is the velocity of the reaction, V is the maximal velocity, Sis the concentration of substrate with Michaelis constant of K_(M), andI is the concentration of inhibitor.

Results are presented as:

-   -   A under 50 nanomolar    -   B 50-500 nanomolar    -   C 501-1000 nanomolar    -   D 1001-5000 nanomolar    -   E 5001-10 000 nanomolar    -   F in excess of 10 000 nanomolar

TABLE 1 Ki Ki Ki Example Test Number cathepsin K cathepsin S cathepsin L1 Test 1 A F D 2 Test 1 A F D 3 Test 1 A F C 1 Test 2 1.6 nM 25 000 nM2000 nM 2 Test 2 1.1 nM 21 000 nM 1700 nM 3 Test 2 4 nM 22 000 nm 1400nM 4 — 2.7 nM 14 500 nM 300 nM 5 — 0.6 nM 2 900 nM 1 170 nm Ref Ex 9 —2.6 nM 6 100 nM NA

The compounds of formula II are thus potent inhibitors of cathepsin Kand yet selective over the closely related cathepsin S and L. Thecompound of reference example 9 provides confidence that thecorresponding difluorovinyl compounds are also active and selective

Metabolic Stability

Compounds of the invention and the indicated comparative examples weretested for metabolic stability in a cytosol assay in which the compoundswere incubated with commercially available human hepatic cytosolfractions and the disappearance of the compound monitored by HPLC orLC/MS. Pooled human liver cytosol fractions are less likely to representoutlier individuals than blood from a single individual and can bestored frozen, unlike whole blood. The cytosol assay thus provides aconsistent assay testbed as a guide to the stability of a compound inthe in vivo environment, such as when exposed to whole blood.

In short, test compounds (2 μM) are incubated in pooled human livercytosol (Xenotech LLC Lenexa US, 1 mg/mL protein in 0.1 M phosphatebuffer, pH 7.4) at 37° centigrade over a one hour period. Theincubations are initiated by the addition of 1 mM NADPH co-factor. Timedsub-samples were taken at 0, 20, 40 and 60 minutes and “crashprecipitated” by the addition of 3 volumes of ice-cold acetonitrile. Thesamples were centrifuged at reduced temperature and the supernatantswere separated and analyzed by LC-MS-MS.

Alternatively, an analogous stability assay is carried out in human ormonkey whole blood and/or commercially available liver microsomes, suchas XEN 025.

TABLE 2 CLint CLint whole blood HLM Example Structure ul/min/mgul/min/mg comparative example

9 49 Example 1

3 16

Comparative Example 1 represents a compound bearing a carbon-carbon bondat the 6 position within the scope of WO2008/007107 cited above. It wasprepared in a facile manner from compound 1d (scheme 1). Hence with theexocyclic alkene 1d in hand, stereoselective hydrogenation of the alkenewith Adams' catalyst (platinum dioxide) in ethyl acetate under ahydrogen atmosphere, proceeded with syn addition of hydrogen. Thishydrogenation afforded essentially one product, namely the C-6 methylisomer (LCMS [M+H]=288 found) with R-stereochemistry in good yield. Thefacial selectivity seen here for the hydrogenation step, is similar tothat reported previously in the literature for a closely relatedbicyclic structure (Srinivas et al, Synlett, 1999, 555-556). The thusprepared building block was deprotected, elongated and oxidised to theactive keto form as for the compounds of the invention exemplifiedabove.

It will be apparent from Comparative Example 1 that a methyl group atthe 6 position provides a compound with whole blood CLint value of 9micrograms/minute/mg, representing an estimated whole blood half life oflittle over an hour. In contrast acetylene provided a Clint value of 3,which represents a calculated whole blood half life approaching 4 hours.Note also that the HLM microsome clearances (representing thecontribution of the liver to metabolism of the respective compounds) issignificantly higher for the 6-methyl species than for the compound ofthe invention, which will further accentuate the better stability of thepresent invention. Improved stability in vivo allows for a betterdistribution of the compound in the body throughout the day,notwithstanding QD or BID dosing. This is particularly important forindications such as osteoporosis where diurnal variation is significant.

Permeability

This experiment measures transport of inhibitors through the cells ofthe human gastroenteric canal. The assay uses the well known Caco-2cells with a passage number between 40 and 60.

Apical to Basolateral Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.5 mL and 0.4 mL transport buffer(TB), respectively, and the standard concentration of the testedsubstances is 10 μM. Furthermore all test solutions and buffers willcontain 1% DMSO. Prior to the experiment the transport plates arepre-coated with culture medium containing 10% serum for 30 minutes toavoid nonspecific binding to plastic material. After 21 to 28 days inculture on filter supports the cells are ready for permeabilityexperiments.

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denotedWash, row 2 “30 minutes” and row 3 “60 minutes”. Transport plate no 2comprises 3 rows of 4 wells, one denoted row 4 “90 minutes”, row 5 “120minutes and the remaining row unassigned.

The culture medium from the apical wells is removed and the inserts aretransferred to a wash row (No. 1) in a transport plate (plate no. 1) outof 2 plates without inserts, which have already been prepared with 1.5mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→Bscreening the TB in basolateral well also contains 1% Bovine SerumAlbumin.

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts and the cell monolayers equilibrated in the transport buffersystem for 30 minutes at 37° C. in a polymix shaker. After beingequilibrated to the buffer system the Transepithelial electricalresistance value (TEER) is measured in each well by an EVOM chop stickinstrument. The TEER values are usually between 400 to 1000Ω per well(depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to the 30 minutes row (No. 2) and fresh 425 μLTB (pH 6.5), including the test substance is added to the apical (donor)well. The plates are incubated in a polymix shaker at 37° C. with a lowshaking velocity of approximately 150 to 300 rpm.

After 30 minutes incubation in row 2 the inserts will be moved to newpre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60minutes), 4 (90 minutes) and 5 (120 minutes).

25 μL samples will be taken from the apical solution after ˜2 minutesand at the end of the experiment. These samples represent donor samplesfrom the start and the end of the experiment.

300 μL will be taken from the basolateral (receiver) wells at eachscheduled time point and the post value of TEER is measured at the endthe experiment. To all collected samples acetonitrile will be added to afinal concentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Basolateral to Apical Transport

Generally every compound will be tested in 2-4 wells. The basolateraland the apical wells will contain 1.55 mL and 0.4 mL TB, respectively,and the standard concentration of the tested substances is 10 μM.Furthermore all test solutions and buffers will contain 1% DMSO. Priorto the experiment the transport plates are precoated with culture mediumcontaining 10% serum for 30 minutes to avoid nonspecific binding toplastic material.

After 21 to 28 days in culture on filter supports the cells are readyfor permeability experiments. The culture medium from the apical wellsare removed and the inserts are transferred to a wash row (No. 1) in anew plate without inserts (Transport plate).

The transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash”and row 3 is the “experimental row”. The transport plate has previouslybeen prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mLTB (pH 7.4), including the test substance, in experimental row No. 3(donor side).

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to theinserts in row No. 1 and the cell monolayers are equilibrated in thetransport buffer system for 30 minutes, 37° C. in a polymix shaker.After being equilibrated to the buffer system the TEER value is measuredin each well by an EVOM chop stick instrument.

The transport buffer (TB, pH 6.5) is removed from the apical side andthe insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is addedto the inserts. After 30 minutes 250 μL is withdrawn from the apical(receiver) well and replaced by fresh transport buffer. Thereafter 250μL samples will be withdrawn and replaced by fresh transport bufferevery 30 minutes until the end of the experiment at 120 minutes, andfinally a post value of TEER is measured at the end of the experiment. A25 μL samples will be taken from the basolateral (donor) compartmentafter ˜2 minutes and at the end of the experiment. These samplesrepresent donor samples from the start and the end of the experiment.

To all collected samples acetonitrile will be added to a finalconcentration of 50% in the samples. The collected samples will bestored at −20° C. until analysis by HPLC or LC-MS.

Calculation

Determination of the cumulative fraction absorbed, FA_(cum), versustime. FA_(cum) is calculated from:

${FA}_{cum} = {\sum\;\frac{C_{RI}}{C_{DI}}}$Where C_(Ri) is the receiver concentration at the end of the interval iand C_(Di) is the donor concentration at the beginning of interval i. Alinear relationship should be obtained.

The determination of permeability coefficients (P_(app), cm/s) arecalculated from:

$P_{app} = \frac{\left( {k \cdot V_{R}} \right)}{\left( {A \cdot 60} \right)}$where k is the transport rate (min⁻¹) defined as the slope obtained bylinear regression of cumulative fraction absorbed (FA_(cum)) as afunction of time (min), V_(R) is the volume in the receiver chamber(mL), and A is the area of the filter (cm²).Reference Compounds

Category of absorption in man Markers % absorption in man PASSIVETRANSPORT Low (0-20%) Mannitol 16 Methotrexate 20 Moderate (21-75%)Acyclovir 30 High (76-100%) Propranolol 90 Caffeine 100 ACTIVE TRANSPORTAmino acid transporter L-Phenylalanine 100 ACTIVE EFFLUX PGP-MDR1Digoxin 30

Greater permeability through the gastrointestinal tissue is advantageousin that it allows for the use of a smaller dose to achieve similarlevels of exposure to a less permeable compound administered in a higherdose. A low dose is advantageous in that minimises the cost of goods fora daily dose, which is a crucial parameter in a drug which is taken forprotracted time periods.

The compound of Example 2 exhibited a p_(app) value of 9.1×10⁻⁶ cm/secin the Caco-2 assay, whereas the prior art compound of Example 2 ofWO2008 007107 exhibited a p_(app) value of 2.7×10⁻⁶ cm/sec in aside-by-side assay run. In this assay system the arguably prior artcompoundN—((S)-1-((3aS,6R,6aS)-6-methoxy-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H,6H,6aH)-yl)-4-methyl-oxopentan-2-yl)-4-(2-(4-methylpiperazine-1-yl)thiazol-4-yl)benzamidewhich is recited at page 33 of WO2008/007107 (but whose preparation isnot disclosed) exhibits a p_(app) value of 0.9×10⁻⁶ cm/sec.

As a rule of thumb, a p_(app) value around 2 represents an in vivoabsorption of only 10-30% whereas a P_(app) value approaching 10 willgenerally represent complete absorption.

The substantial difference in p_(app) values between Example 2 and theabovementioned prior art Example 2 of WO2008 007107 correlates well within vivo mouse PK experiments, as illustrated in FIG. 1. The respectivecompounds were orally administered (60 μmol/kg in a conventional 1%Methocel A4C vehicle. As seen clearly in FIG. 1, the in vivo exposure(whether measured as C_(max) or AUC) was very much greater for thecompound of the invention than the prior art compound (Example 2 ofWO2008 007107). Note that the graph has a logarithmic scale

Mutagenicity

The mutagenic potential of compounds is conveniently tested in the AmesTest, typically carried out in a variety of bacterial strains such asSalmonella typhimurium TA100, TA102, TA 1535, TA 1537 with and withoutliver S9 fraction activation, for example at 30, 300 and 3000 ug/plateconcentrations.

Ames testing is readily available at a number of CROs around the world.

ABBREVIATIONS

DMF dimethylformamide DCM dichloromethane TBDMS tert-butyldimethylsilylRT room temperature THF tetrahydrofuran Ac acetyl TLC thin layerchromatography DMAP dimethylaminopyridine EtOAc ethyl acetate uMmicromolar

All references referred to in this application, including patents andpatent applications, are incorporated herein by reference to the fullestextent possible.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

The invention claimed is:
 1. A compound of the formula:

wherein the —C═C(R1)R2 moiety is acetylene, and the N-function isoptionally protected with an N-protecting group selected from the groupconsisting of formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, benzenesulfonyl,p-toluenesulfonyl, benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, benzyl(bz), triphenylmethyl, benzyloxymethyl trimethylsilyl, phenylsulfonyl,t-butoxycarbonyl (BOC), and benzyloxycarbonyl (Cbz).
 2. A compound ofthe formula:

wherein the N-function is optionally protected with an N-protectinggroup selected from the group consisting of formyl, acetyl, propionyl,pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl,benzenesulfonyl, p-toluenesulfonyl, benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, benzyl(bz), triphenylmethyl, benzyloxymethyl trimethylsilyl, phenylsulfonyl,t-butoxycarbonyl (BOC), and benzyloxycarbonyl (Cbz).
 3. A compound ofthe formula:


4. A compound of the formula

or of the formula

wherein the —C═C(R1)R2 moiety is acetylene and wherein the N-function isprotected with a Boc, CBz or Fmoc group.
 5. A compound according toclaim 4, wherein the N-function is protected with a Boc group.
 6. Aprocess for the preparation of a compound of formula 1h comprising thesteps of converting the aldehyde group of a compound of formula 1f to anacetylene group of a compound of formula 1h effected by i) treatment ofthe aldehyde of formula 1f with triphenylphosphine and carbontetrabromide, thus forming the dibromoalkene of formula 1g, and ii)treatment of the dibromoalkene of formula 1g with butyllithium, thusproviding the acetylene derivative of formula 1h, as outlined in thescheme below:


7. The process according to claim 6, wherein the compound of formula 1fis prepared by oxidation of the alcohol of a compound of formula 1e(6-Hydroxymethyl-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylicacid tert-butyl ester) effected by treatment with Dess Martinperiodinane in DCM, as outlined in the scheme below:


8. The process according to claim 7, wherein the compound of formula 1eis prepared by (a) oxidation of the compound of formula 1c effected bytreatment with Dess Martin periodinane in DCM, to a keto compound asoutlined in the step below:

(b) transformation of the keto compound of step (a) to an exoolefin offormula 1d effected by treatment with Dess Martin periodinane in DCM andmethyl triphenylphosphoniumbromide, potassium t-butoxide, and THF, asoutlined in the step below:

(c) hydroxylation of the compound of formula 1d to a compound of formula1e effected by treatment with 9-borabicyclo[3.3.1]nonane (9-BBN—H) andTHF, as outlined in the step below: